Xiaoyao Kangai Jieyu Fang, a Chinese Herbal Formulation, Ameliorates Cancer-Related Depression Concurrent with Breast Cancer in Mice via Promoting Hippocampal Synaptic Plasticity

Diagnosis with breast cancer is a major life event that elicits increases in depressive symptoms for up to 50% of women. Xiaoyao Kangai Jieyu Fang (XYKAJY) is derived from a canonical TCM formula, Xiaoyao San (XYS), which has a history of nearly 1000 years for treating depression. The aim of this study was to investigate whether XYKAJY alleviates depression-like behavior and breast tumor proliferation in breast cancer mice then explore the mechanisms underlying its action on HPA axis and hippocampal plasticity further. XYKAJY was treated at the high dose of 1.95 g/mL and 0.488 g/mL, after 21 days of administration. Different behaviors, monoamine neurotransmitters, tumor markers, and the index of HPA axis were detected to evaluate depressive-like symptoms of breast cancer mice. Also, the pathological changes of the tumor, hippocampus, and the expressions of GR, NR2A, NR2B, CAMKII, CREB, and BDNF were detected. In this study, XYKAJY formulation significantly improved the autonomic behavior, reduced the incubation period of feeding, and reversed the typical depressive-like symptoms in breast cancer mice. Also, it reduced the content of CORT, ACTH, CRH, and CA125, CA153, CEA in the blood, protected the pathological changes of the hippocampus and tumor, upregulated the expression of GR, CREB, and BDNF in the hippocampus, and significantly decreased the expression of NR2A, NR2B, and CaMKII. These results provide direct evidence that XYKAJY effectively alleviates depression-like behaviors and tumor proliferation in vehicle mice with ameliorates hippocampus synaptic plasticity dysfunctions

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

<div><p>Background</p><p>The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).</p> <p>Methodology/Principal Findings</p><p>The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The <i>in vivo</i> protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior <i>in vivo</i> protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.</p> <p>Conclusions/Significance</p><p>Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.</p> </div

Analysis of mAbs against HAs of clade 0, 1, and 2 H5N1 viruses.

<p>(A) Amino acid sequences of VH and VL region and their closest corresponding human Ig gene. (B) Immunofluorescent analysis of mAbs. The MDCK cells were infected with H1N1 influenza virus A/Puerto Rico/8/34 (PR8) or recombinant influenza viruses carrying HA from clade 0, 1, and 2 H5N1 viruses in r97HKHA-PR8, r04VNHA-PR8, and r06ZJHA-PR8. (C) Western blot analysis. The HA proteins expressed in MDCK cells infected with r97HKHA-PR8, r04VNHA-PR8, r06ZJHA-PR8 or A/Puerto Rico/8/34 were used to determine the binding of the mAbs. Arrows indicate the specific bands for the corresponding proteins.</p

Analysis of lungs from mice treated with mAb 4E6 and 1H10 after infection with H5N1 virus A/Vietnam/1194/04.

<p>(A) Viral copy number in the lungs at day 6 post-infection was determined using Q-PCR. The results are expressed as the mean ± SEM. (B) Virus titer in the lungs at day 6 post-infection was measured using a plaque formation assay in MDCK cells. The results are expressed as the mean ± SEM. (C) Histopathology in pulmonary tissue (40x magnification). 1) Mouse treated with mAb 4E6 at 24 hours post-infection. 2) Mouse treated with mAb 1H10 at 24 hours post-infection. 3) Mice injected with an irrelevant IgG at 24 hours post-infection were used as the control. HE: H&E staining. IHC: immunohistochemical staining using a mAb against influenza nucleoprotein.</p

Therapeutic efficacy of mAb 4E6 and 1H10 in a mouse model of H5N1 virus A/Vietnam/1194/04 infection.

<p>(A) The Kaplan-Meier survival curve. BALB/c mice (n=7 per group in two separate experiments) were infected intranasally with 10 LD₅₀ of A/Vietnam/1194/04 and at 24, 48, or 72 hours, were treated with a single intraperitoneal injection of mAb 4E6 or 1H10 at 10 mg/kg body weight. The control group received IgG at 10 mg/kg body weight at 24 hours post-infection. (B) Percentage weight change in mice treated with mAb 4E6 or 1H10 at different time points post-infection. The mice (n=7) were infected via intranasal inoculation with 10 LD50 of A/Vietnam/1203/4, followed by a single intraperitoneal injection of mAbs at 24, 48, or 72 hours post-infection, and their weights were monitored for 14 days. The mice injected with an irrelevant IgG at 24 hours post-infection were used as the control group.</p

Schematic presentation depicting the method and process to generate human-like neutralizing mAbs through immunization of rhesus macaques.

<p>Schematic presentation depicting the method and process to generate human-like neutralizing mAbs through immunization of rhesus macaques.</p

Recombinant adenoviruses carrying codon-optimized HA genes expressed HA proteins and elicited neutralizing antibodies against H5N1 viruses in rhesus macaques.

<p>(A) Schematic representation of adenoviral vectors carrying H5N1 influenza HA gene (Ad5-HA). The expression cassette, containing the HA gene under the control of the CMV promoter and BGH polyA signal, was cloned into the adenoviral E1 region. (B) Expression of the HA protein mediated by recombinant adenovirus Ad5-HAs. The HA proteins before cleavage (H0) and after cleavage (HA1 and HA2) were detected using chicken serum against HA in Western blots. The Vero cells were harvested for analysis at 48 hours after infection with Ad-Empty, Ad5-HA04VN, Ad5-HA97HK, and Ad5-HA06ZJ at MOIs of 10. (C) Dynamics of the antibody response in a Chinese rhesus macaque immunized with Ad5-HAs. The sera were collected at 2-week intervals. The neutralizing titers against the recombinant H5N1 viruses (r97HKHA-PR8, r04VNHA-PR8, and r06ZJHA-PR8) were determined using a micro-neutralization assay. The arrows indicate the time of immunization. (D) The binding specificity of the sera against HA of different clades expressed by the recombinant influenza viruses was determined by Western blot analysis.</p

Binding kinetics of mAbs to HA protein.

<p>Binding kinetics of mAbs to HA protein.</p