A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

[Genetic epidemiologic study on nasal polyps].

OBJECTIVE: To investigate the genetic mode of nasal polyp and the effect of genetic factor on occurrence of the disease. METHODS: A genetic epidemiological case-control study including 280 pedigrees (120 nasal polyp cases and 160 controls) was conducted. The segregation ratio and the heritability of nasal polyp were respectively estimated by the Li-Mantel-Gart method and the Falconer method. RESULTS: The segregation ratio was 0.124 (95% CI 0.081-0.167), significantly lower than 0.25, which showed that nasal polyp did not possess the characteristics of monogenetic model. The prevalence rate of first-degree and second-degree relatives in cases were 8.571% and 3.086% respectively, which were significantly different (X2 = 24.851, P < 0.01) and were higher than that noticed 1.376% and 1.141% in controls (X2 = 33.547 and 14.274, all P < 0.01). The heritability of the first-degree and second-degree relatives of nasal polyp was 64.488% and 61.947%. Among the first-degree relatives of nasal polyp probands, the heritability of the adult group and the children group were respectively 60.735% and 74.598% (the difference was significant, X2 = 4.504, P < 0.05). The heritability of the first-occurred group was 62.839% and the recurred group was 74.304% (the difference was significant, X2 = 4.105, P < 0.05). CONCLUSIONS: This study indicated that the genetic model of nasal polyp belonged to polygenetic and the genetic factors played an important role in the occurrence of nasal polyp, especially for young or recurred patients.

[Real-time PCR used to detect p53 gene damage in workers exposed to arsenic].

OBJECTIVE: To evaluate the relationship between the metabolism of arsenic and the damage of exon 5 and 8 of p53 gene from workers in a arsenic mill, and with real-time PCR technique, to establish the method probing the gene-specific DNA damage in people. METHODS: By real-time PCR, the damages of exon 5 and 8 of p53 gene were probed in 37 workers exposed highly to, 16 manager and logistic employees exposed less to an arsenic mill in Yunnan province, and also 25 local people who did not contact with any white arsenic in near past time. At the same time, the urinary total and organic arsenic of workers were detected. The correlation between metabolism of arsenic and damage of p53 gene was evaluated. RESULTS: Total urinary arsenic concentrations were (1.18 +/- 0.76) mg/L and (0.32 +/- 0.28) mg/L for high and low exposed male workers, and 0.23 mg/L, (0.53 +/- 0. 30) mg/L for high and low exposed females. Organic urinary arsenic concentrations were (0.48 +/- 0.37) mg/L and (0.08 +/- 0.05) mg/L for high and low exposed males, and 0.11 mg/L, (0.30 +/- 0.24) mg/L for high and low exposed females. The total and organic urinary arsenic of high exposed group was higher than that of control male (P < 0.05), all in control group were lower than 0.02 mg/L for reference. The Ct relative value of exon 5 of p53 gene in high exposed group was higher than that in control male (P < 0.05), and the increased tendency of Ct relative value of exon 5 of p53 gene was found in workers with organic arsenic concentration going up (r(s) = 0.355, P = 0.011). The Ct relative value of exon 8 of p53 gene in low exposed group was higher than that in control male (P < 0.05), but the difference between high exposed and low exposed or reference's was not obvious (P > 0.05). CONCLUSION: The damages in exon 5 and 8 of p53 gene in workers exposed to arsenic may be induced. The metabolism of arsenic may be very important in the damage of exon 5. It is feasible for real-time PCR technique used to detect gene-specific DNA damage in people.

[Study on the association between urinary organic arsenic and 8-hydroxydeoxyguanine in workers exposed to arsenic].

OBJECTIVE: To evaluate the association between metabolism of arsenic and DNA oxidative damage in workers in a arsenic mill. METHODS: Urinary organic arsenic and 8-hydroxydeoxyguanine were detected in 37 workers highly exposed to arsenic and 16 administrative and logistic staff with mild exposure in a arsenic mill in Yunnan province, and also 28 local people who did not have the exposure in the near past time. The correlation between metabolism of arsenic and DNA oxidative damage was evaluated. RESULTS: The urinary organic arsenic concentration was respectively (0.48 +/- 0.37) mg/L and (0.08 +/- 0.05) mg/L for men with high and low exposure, and was respectively 0.11 mg/L and (0.30 +/- 0.24) mg/ L for women with high and low exposure, while it was lower than 0.02 mg/L in the controls. Urinary 8-hydroxydeoxyguanine concentration was (18.07 +/- 11.68) micromol/mol creatinine, (11.79 +/- 8.25) micromol/mol creatinine, (10.07 +/- 3.04) micromol/mol creatinine for the males with high and low exposure and of controls, respectively, (P < 0.05), and it was 84.35 micromol/mol creatinine, (21.27 +/- 5.89) micromol/mol creatinine, (14.43 +/- 2.58) micromol/mol creatinine for females with high and low exposure and of controls, respectively. The female workers exposed to arsenic had higher urinary 8-hydroxydeoxyguanine levels than males did (P < 0.05). The increased tendencies of urinary 8-hydroxydeoxyguanine levels with the organic arsenic concentration were found in workers (r(s) = 0.279, P = 0.019). CONCLUSION: Occupational individuals exposed to arsenic have obvious DNA oxidative damage, which is more severe in females. The difference of metabolism of arsenic may play a key role.

[Experiment research on the location of DNA lesion in the nucleic acid sequence].

OBJECTIVE: To research on establishing a method that can detect the DNA lesion at the level of nucleic acid. METHODS: The TK6 cell was cultured and treated, the genomic DNA was extracted, and the strand cleavage was induced by the endonuclease Hinf I. Then the genomic DNA was amplified by randomized terminal linker-dependent PCR (RDPCR), and hybridized by Southern hybridization with the single-stranded probes of the exon 2 of k-ras gene. The PCR products were sequenced. RESULTS: The clear hybridized band from the products of RDPCR was seen at the expectant position cut by Hinf I. The sequencing analysis showed that the position of DNA lesion linked by the linker was just the restriction site of Hinf I. CONCLUSION: It can be used to detect the DNA lesion at the level of nucleic acid sequence with the combination of sequence and RDPCR technologies.

[Effects of mixed cypermethrin and methylparathion on endocrine hormone levels and immune functions in rats: II. Interaction].

OBJECTIVE: To study interaction of mixed pesticides cypermethrin and methyl parathion on reproductive hormones, thyroid hormones, and immune functions in rats. METHODS: Eighty 2-month old Wistar rats (40 male and 40 female) were divided randomly by body weight into 8 groups. The dose 1/30 LD50 were chosen for the single or combined exposure representing respective doses of 0, cypermethrin 8.0 mg/kg bw, methylparathion 0.23 mg/kg bw, and 1/30 LD50 cypermethrin plus 1/30 LD50 methylparathion. The control group received vehicle solvent only. All groups were force-fed every two days for 30 days. Body weight gain and organ weights were determined. Serum levels of IgG and IgA, reproductive hormones [luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2), and testosterone], as well as the thyroid hormones [triiodothyronine (T3), tetraiodothyronine (T4), and thyroid stimulating hormone (TSH) were measured using radioimmunoassay (RIA). In addition, two immunological parameters (rate of neutrophil phagocytosis, rate of lymphocyte transformation] were being measured in blood samples. RESULTS: The most of index indicated addictive interaction, while the effects on relative weights of ovaries and adrenals, IgA and rate of lymphocyte transformation were antagonistic. It was of interest that the effect on estradiol was synergistic interaction in female rats, whereas it was addictive interaction in male rats, whose estradiol level could be increased 64.64% by cypermethrin exposure. CONCLUSION: Our results showed that exposure to cypermethrin and methyl parathion mixture at 1/30 LD50 dose had interaction on endocrine hormone levels, and immune functions in rats. Estradiol was very sensitive, the mixture can enhance estradiol level both in male and female rats.

[Effects of mixed cypermethrin and methylparathion on endocrine hormone levels and immune functions in rats: I. Dose-response relationship].

OBJECTIVE: To study dose-response relationship effects of mixed cypermethrin and methyl parathion on reproductive hormones, thyroid hormones, and immune functions in rats. METHODS: Eighty 2-month old Wistar rats (40 males and 40 females) were divided randomly by bodyweight into 4 groups. Four doses (0, 1/600 LD50, 1/135 LD50 and 1/30 LD50) were chosen for the combined exposure representing respective doses of cypermethrin 0, 0.4, 1.8 and 8.0 mg/kg body weight and of methylparathion 0, 0.0115, 0.0518 and 0.2300 mg/kg body weight. The control group received vehicle solvent only. All groups were force-fed every two days for 30 days with these dose combinations. Body weight gain and organ weights were determined. Serum levels of IgG and IgA, reproductive hormones (luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2), and testosterone), as well as the thyroid hormones (triiodothyronine (T3), tetraiodothyronine (T4), and thyroid stimulating hormone (TSH) were measured using radioimmunoassay (RIA). In addition, two immunological parameters (rate of neutrophil phagocytosis, rate of lymphocyte transformation) were being measured in blood samples. RESULTS: The body weight gains were similar in all 4 groups. The weights of adrenal glands in exposed rats were heavier than those in control (P < 0.05). Serum FSH and E2 levels in exposed rats were higher than those in the control group (P < 0.01). Serum TSH levels were proportionally increasing with higher pesticide doses (r(s) = 0.329, P < 0.01). Lymphocyte transformation rates in all exposed animals were lower than that of the control group (P < 0.01). To the contrary, rates of neutrophil phagocytosis in all exposure groups were higher than those of the control group (P < 0.01). Furthermore, serum IgG levels of all exposed animals were lower than that of the control (P < 0.01) and serum IgA levels in exposed females were higher than that of the control (P < 0.01). Dose-response relationships for these changes were significant (rank correlation statistics P < 0.05 or < 0.01). CONCLUSION: Our results showed that exposure to different mixtures of cypermethrin and methyl parathion disrupted the endocrine hormone levels, and immune functions in rats.