Bovine FcRn-Mediated Human Immunoglobulin G Transfer across the Milk-Blood Barrier in Transgenic Mice

<div><p>Maternal-fetal IgGs transport occurs either prenatally or postnatally, which confers the newborns with passive immunity before their own immune system has matured. However, little is known about the mechanisms of postnatal IgGs passage in the mammary gland. To investigate how FcRn mediates the IgGs transport in the mammary gland, we first generated bFcRn and anti-HAV mAb transgenic mice, and then obtained HF transgenic mice expressing both transgenes by mating the above two strains. Transgene expression of bFcRn in the four lines was determined by qRT-PCR and western blot. We then localized the expression of bFcRn to the acinar epithelial cells in the mammary gland, and anti-HAV mAb was mainly detected in the acini with weak staining in the acinar epithelial cells. Human IgGs could be detected in both milk and serum of HF transgenic mice by western blot and ELISA. A significantly lower milk to serum ratio of human IgGs in HF mice compared with that of anti-HAV mAb mice, indicating that bFcRn could transport human IgGs across the milk-blood barrier from milk to serum during lactation in HF mice. While, there were no transport of murine IgGs, IgAs, or IgMs. These results provide understandings about the mechanisms of maternal-fetal immunity transfer in the mammary gland.</p></div

Is Adjuvant Cellular Immunotherapy Essential after TACE-Predominant Minimally-Invasive Treatment for Hepatocellular Carcinoma? A Systematic Meta-Analysis of Studies Including 1774 Patients

<div><p>Purpose</p><p>Cellular immunotherapy has appeared to be a promising modality for the treatment of malignant tumor. The objective of this study was to evaluate the efficacy of cellular immunotherapy combined with minimally invasive therapy.</p><p>Methods</p><p>We searched PubMed, Web of Science and The Cochrane Library through March 2016 for relevant studies. Short-term efficacy (the disease control rate, the control rate of quality life and the AFP descent rate) and long-term efficacy (overall survival (OS) and progression-free survival (PFS) rate) were compared as the major outcome measures. The meta-analysis was performed using Review Manager 5.3.</p><p>Results</p><p>A total of 1174 references in 3 databases were found of which 19 individual studies with 1774 HCC patients enrolled in this meta-analysis. Meta-analysis results showed that cellular immunotherapy combined with minimally-invasive treatment significantly improved the measures of short-term response (the disease control rate (OR = 5.91, P = 0.007), the control rate of quality lift (OR = 3.38, P = 0.003) and the AFP descent rate (OR = 4.48, P = 0.02)). Also higher 6-month PFS (OR = 2.78, P = 0.05), ≥12-month PFS (OR = 3.56, P<0.00001) rate and 6-month OS (OR = 2.81, P = 0.0009), 12-month OS (OR = 3.05, P<0.00001) and 24-month OS (OR = 3.52, P<0.0001) rate were observed in patients undergoing cellular immunotherapy.</p><p>Conclusions</p><p>This meta-analysis suggested that cellular immunotherapy is a feasible adjuvant treatment that could be beneficial for the improvement of the clinical outcomes for hepatocellular carcinoma (HCC) patients after minimally invasive treatment, including short-term response and long-term survival.</p></div

bFcRn cannot transport murine IgAs and IgMs across the mammary gland.

<p>bFcRn could neither increase the concentration of IgAs and IgMs (A and C) nor transport them across the milk-blood barrier in the mammary gland (B and D). The ratio of serum/milk indicates the transfer of IgAs or IgMs from serum to milk. The data were combined from three independent experiments; the bars represent the means ± SD (n = 7–8 mice per group).</p

ELISA analysis of human IgGs in the mammary gland of transgenic mice.

<p>Human IgGs were detected in the serum of HF mice (A–B) and the milk/serum ratio was significant lower in HF transgenic mice (C–D). The milk/serum ratio is used as an index for the transfer of human IgGs from milk to serum in the mammary gland. The concentrations of murine IgGs (E–F) and the ratio of serum/milk (G–H) indicated that there was no transfer of murine IgGs by bFcRn. The ratio of serum/milk is used as an index for the transfer of murine IgGs from serum to milk. D3-serum, serum obtained on day 3 of lactation; C, colostrum collected on day 3 of lactation; D10-serum, serum obtained on day 10 of lactation; M, milk collected on day 10 of lactation. The data were combined from three independent experiments; the bars represent the means ± SD (n = 7–8 mice per group); <i>*P</i><0.05; <i>**P</i><0.01.</p

Generation of the bFcRn and anti-HAV mAb transgenic mice.

<p>(A–B) Schematic representation of the bFcRn and anti-HAV mAb constructs for microinjection. Chicken β-globin insulator (2x), un-translated exons E1, E8, E9, parts of E2 and E7, and also β-casein 3′ genomic DNA are indicated by bars. Goat β-casein promoter is indicated by solid arrow. The restriction sites of <i>Sal</i>I, <i>Xho</i>I, <i>Not</i>I, <i>Eco</i>RI, <i>Noc</i>I are shown. The hatched boxes represent insertion of sequences of <i>bFcRn α, bFcRn β, human IgG HC,</i> and <i>human IgG LC</i> to <i>Xho</i>I restriction site, respectively. Double arrows indicate the probes used for the southern blot for <i>bFcRn</i> and <i>anti-HAV mAb</i> transgenes. Arrows in (A) represent the quantitative real-time PCR primers used for the detection of bFcRn α. (C–D) Identify transgenic founders using PCR. M, 1 kb DNA ladder in (C) or 100 bp DNA ladder in (D); P1, P2, positive plasmids control for <i>bFcRn α</i>, <i>bFcRn β,</i> respectively; P, positive plasmids control for <i>human IgG HC</i> or <i>human IgG LC</i>; Neg, genomic DNA from wild-type mice as a negative control. (E–F) Southern blot analysis of transgenes. P10, plasmid control of ten copies of <i>bFcRn α</i> or <i>bFcRn β</i>; P1, P5, P10, plasmids control equivalents of 1, 5, 10 copies of <i>human IgG HC</i> or <i>human IgG LC</i>; Neg, genomic DNA from an age matched wild-type mouse as a negative control.</p

Comparison of short-term efficacy between the patients undergoing cellular immunotherapy or not by using the random effects model (Mantel-Haenszel method).

<p>Comparison of short-term efficacy between the patients undergoing cellular immunotherapy or not by using the random effects model (Mantel-Haenszel method).</p

Analysis of the distribution of human IgGs in anti-HAV mAb and HF transgenic mice.

<p>Human IgGs distribution in the anti-HAV mAb transgenic mice (A) and co-localization of bFcRn and human IgGs in the HF transgenic mice (B). Tissue sections prepared from the mammary gland of anti-HAV mAb mice were incubated with Cy3-conjugated (red) goat anti-human IgG antibody. For the HF mice, the sections were hybridized with a combination of Cy3-conjugated (red) goat anti-human IgG antibody and FITC-conjugated anti-bFcRn α antibody (K6). The nucleus was stained with DAPI (blue). The data obtained are representative of at least three sections. Images were obtained with a 40X water objective lens in (A) and a 100X oil objective lens in (B). (C) Human IgGs were transferred from milk to serum in the HF transgenic mice. Milk and serum (day3) were performed western blot using goat anti-human IgG antibody in four different genotypes of mice from line-1. S, serum; M, milk; P, positive control, human IgG.</p

Flow chart of the included studies.

<p>Flow chart of the included studies.</p

Funnel plots to detect any publication bias.

<p>The P value located in this figure indicates the results of Peters test for assessment of publication bias.</p

Comparison of 6, 12, 24-month OS between the patients undergoing cellular immunotherapy or not.

<p>The random effects model (Mantel-Haenszel method) was used.</p