Advance care planning for frail older people in China: A discussion paper.

As the aging population, including frail older people, continues to grow in Mainland China, quality of life and end-of-life care for frail older people has attracted much attention. Advance care planning is an effective way to improve end-of-life care for people with advanced diseases, and it is widely used in developed countries; however, it is a new concept in Mainland China. The effects of advance care planning and its acceptability in Mainland China are uncertain because of its culture-sensitive characteristics. The objective of this article is to discuss the serious social issue of caring for frail older people and illustrate the possibility of implementing advance care planning in nursing homes in Mainland China through a review of relevant literature, which will focus on legislation, healthcare system engagement, public engagement, and cultural issues. Recommendations to promote and implement advance care planning include choosing nursing homes as a proper setting, establishing an ethical climate, and enhancing public awareness.

Associated changes in the transcription levels of IL-17A and tight junction-associated genes in the duodenal mucosa of rhesus macaques repeatedly exposed to simian/human immunodeficiency virus.

BACKGROUND: Mucosal barrier dysfunction might play a key role in HIV/AIDS, yet the early effects of HIV-1 on intestinal mucosal barrier, especially tight junctions (TJ) have not been well addressed. AIMS: To investigate the effects of acute HIV-1 infection on the expression of intestinal IL-17A and TJ-associated genes using an NHP-AIDS model. METHODS: TaqMan probe real-time RT-PCR methods were established and claudin-1, claudin-3, occludin and zonula occluden-1 (ZO-1) mRNA levels in the duodenal biopsies of rhesus macaques collected before and after rectal exposures to SHIV-SF162P4 were examined and compared with that of IL-17A, IL-6, TGF-ß, RORγt, T-bet, Foxp-3 and GATA-3. RESULTS: The mRNA levels of TJ-associated genes were statistically significantly reduced soon after viral exposures and the mRNA levels of claudin-1, occludin and ZO-1 in viral positive tissues (from Group I) were lower than that in viral negative tissues (from Group II) after viral exposure. IL-17A mRNA levels were also decreased and positively correlated with the mRNA levels of the TJ-associated genes after viral exposure or infection, although the levels of IL-6, TGF-ß and RORγt mRNA showed no statistical difference. The levels of GATA-3 mRNA in tissues collected before viral exposure were statistically different between Group I and Group II animals. The balance between T-bet and GATA-3 mRNA levels in Group II was markedly altered and statistically significantly different from that in Group I. CONCLUSIONS: Acute SHIV, and by extension HIV infection could affect the expression of TJ-associated genes, probably through IL-17A and other immune alterations.

Mucosal addressin cell adhesion molecule-1 of rhesus macaques: molecular cloning, expression, and alteration after viral infection.

BACKGROUND: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a member of the immunoglobulin superfamily, is essential for gut-specific homing of leukocytes; however, it has not been well characterized in rhesus macaques. AIMS: To obtain the complete nucleotide sequence of rhesus macaque MAdCAM-1 cDNA and determine its distribution in gut-associated lymphoid tissues (GALT) and its alteration in duodenal mucosa after simian/human immunodeficiency virus (SHIV) infection. METHODS: MAdCAM-1 cDNA was cloned from the colon mucosa of a rhesus macaque by 3'- and 5'-RACE. The distribution and abundance of MAdCAM-1 mRNA in the GALT were examined by nested and real-time RT-PCR. The alterations of MAdCAM-1 mRNA levels in SHIV-infected duodenal mucosa were determined by real-time RT-PCR. RESULTS: The nucleotide sequence of rhesus macaque MAdCAM-1 cDNA (1,503 bp nucleotides) including the 5'- and 3'-untranslated regions was obtained. The coding region (1,086 bp) showed 87.56% and the Ig-like domain 1, 2 and TM + cytoplasmic domains showed >93% nucleotide sequence identity to that of humans. Like humans, rhesus macaques lacked MAdCAM-1 IgA1-like domain, which could be a common feature for all primates appeared later during vertebrate evolution. Two species of MAdCAM-1 mRNA were detected and high-level transcripts were observed primarily in the GALT. The full-length MAdCAM-1 expressed in vitro could bind to human α4ß7. MAdCAM-1 mRNA levels were statistically significantly reduced in SHIV-infected duodenal mucosa. CONCLUSIONS: These data provided a basis for using rhesus macaques in pathological and therapeutic studies on leukocyte homing related diseases such as inflammatory bowel disease and HIV/AIDS.

Immunization with recombinant macaque major histocompatibility complex class I and II and human immunodeficiency virus gp140 inhibits simian-human immunodeficiency virus infection in macaques.

Genetic, epidemiological and experimental evidence suggest that the major histocompatibility complex (MHC) is critical in controlling human immunodeficiency virus (HIV) infection. The objectives of this study were to determine whether novel recombinant Mamu MHC constructs would elicit protection against rectal challenge with heterologous simian-human immunodeficiency virus (SHIV) strain SF162.P4 in rhesus macaques. Mamu class I and II gene products were linked together with HIV gp140, simian immunodeficiency virus (SIV) p27 and heat-shock protein 70 to dextran. The vaccine was administered to two groups, each consisting of nine macaques, either subcutaneously (SC), or rectally and boosted by SC immunization. The controls were untreated or adjuvant-treated animals. Repetitive rectal challenges with up to ten doses of SHIV SF162.P4 showed a significant decrease in the peak and sequential viral RNA concentrations, and three macaques remained uninfected, in the nine SC-immunized animals, compared with infection in all nine controls. Macaques immunized rectally followed by SC boosters showed a less significant decrease in both sequential and peak viral loads compared with the SC-immunized animals, and all were infected following rectal challenge with SHIV SF162.P4. Plasma and mucosal IgG and IgA antibodies to Mamu class I alleles and HIV gp120, as well as to RANTES (regulated upon activation, normal T-cell expressed, and secreted; CCR5) were increased, and showed significant inverse correlations with the peak viral load. These results suggested that allo-immunization with recombinant MHC constructs linked to HIV-SIV antigens merits further investigation in preventing HIV-1 infection.

Cellular bases for interactions between immunocytes and enteroendocrine cells in the intestinal mucosal barrier of rhesus macaques.

The roles of the interactions between nervous, endocrine, and immune systems have been well established in human health and diseases. At present, little is known about the cellular bases for neural-endocrine-immune networks in the gastrointestinal mucosa. In the current study, duodenum, jejunum, ileum, cecum, colon, and rectum autopsies from 15 rhesus macaques and endoscopic duodenal biopsies from 12 rhesus macaques were collected, and the spatial relationships between the endocrine cells and immune cells in the intestinal mucosa were examined by transmission electron microscopy. Eight types of enteroendocrine cells similar to human enterochromaffin cells (EC), D1, G, I, K, L, N, and S cells were found to lie within a one-cell-size distance from immunocytes, in particular the eosinophils in the epithelia or lamina propria. Close apposition of large areas of plasma membranes between many types of enteroendocrine cells and immunocytes, especially between EC, K, S cells and eosinophils, were observed in the epithelia for the first time. These data indicate that complex interactions occur between diverse types of enteroendocrine cells and various immune cells through paracrine mechanisms or via mechanisms dependent on cell-to-cell contact; such interactions might play key roles in maintaining the gut mucosal barrier integrity of rhesus macaques.

Elevated frequency of CD1c+ myeloid dendritic cells in the peripheral blood mononuclear cells of simian/human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV) repeatedly infected Chinese rhesus macaques.

CD1c+ myeloid dendritic cells (mDCs) in the peripheral blood of 30 SHIV-SF162p4 and SIVmac251 sequentially infected Chinese rhesus macaques were examined by flow cytometry to obtain further insight into mDC alterations in HIV/AIDS. The CD1c+ cells were found to be mononuclear leukocytes rather than granulocytes, and most of them expressed CD20. CD1c+mDCs (CD1c+CD20-) consisted of two morphological subsets: the granular and the large CD1c+mDCs. The expression of HLA-DR, CD86, and CD11b, but no CCR7, CD83 and CD123, together with their endocytotic capacity indicated that they were immature mDCs. Their frequency at weeks 10 and 12 post-infection was significantly higher than that of un-infected ones; the large CD1c+mDC level was significantly different between time points and almost absent from un-infected rhesus monkeys; significant correlations between CD1c+mDCs and plasma viral load levels were also observed. These data indicated a possible role for CD1c+mDCs in the pathophysiological process of SIV/HIV infection.

mED2--a novel gene involved in mouse embryonic development.

Dissection of new genes underlying embryonic development is important for our understanding of the molecular mechanism of vertebrate embryonic development. In this study, the expression pattern and functional analysis of a new gene, called mED2, originally cloned from mouse embryos using subtractive hybridization was reported. mED2 expression patterns were characterized by RT-PCR-Southern hybridization and in situ hybridization. The results showed that mED2 was mainly expressed in the embryonic nervous system and mesoderm-derived tissues and its expression varied depending on the embryonic developmental stages. The knockdown of mED2 activity by antisense RNA injection inhibited zygote cleavage and blastocyst formation during pre-implantation in mice. Subcellular localization of mED2-eGFP fusion protein revealed a pattern of nuclear membrane and juxta-/perinuclear location such as in the rough endoplasmic reticulum and Golgi apparatus. This finding was supported by bioinformatics analysis, which indicated mED2 protein to be a transmembrane protein with partial homology to the thioredoxin family of proteins. It is inferred that mED2 gene can probably take part in early embryonic development in mouse and may be involved in target protein posttranslational modification, turnover, folding, and stability at the endoplasmic reticulum and/or the Golgi apparatus.

[Transgenic mice produced by exchromosomal homologous recombination combined with cytoplasmic injection].

An expression construct specified in mammary gland with double cistrons of human G-CSF gene and IRES-EGFP gene under control of ovine beta-lactoglobulin gene flanking sequence, has been constructed in two parts (named fragment I and fragment II) that share an overlapping region of 2.2 kb sequence. Two sites of loxp and lox2272 for homologous recombination were inserted into both flanking regions of G-CSF. The lengths of fragment I and fragment II are 5.9 kb and 5.6 kb, respectively. The whole length of the expression vector (beta-LG-hG-CSF-IRES-EGFP) is 9.3 kb. The two DNA fragments mixed with nuclear locating sequence DNA (NLS) fragment were coinjected into murine zygote cytoplasm. The resulting mice were analyzed for the transgene integration and expression. A total of 138 founders were born. 62.3% (86/138) of them was integrated with fragment I sequence, and 54.3% (75/138) of them contained fragment II, whereas 62 of them contained both fragment I and II, of whom 80. 6% (50/62) were integrated by the whole reconstituted gene construct (result of ex-chromosomal homologous recombination, ECR). By RT-PCR analysis, it was shown that 90% of the ECR mice (9/10) expressed human G-CSF gene and 100% expressed EGFP gene. EGFP expression was also detected by absorption spectrum scanning from 400 nm to 700 nm, and 50% (5/10) of the ECR mice expressed EGFP protein. The high frequency and accuracy of homologous recombination in murine zygotes reported here suggests that some large transgenes could be constructed by ECR pathway. This method does not need to construct a long complex DNA vector directly and to do nucleus-injection, and is easier than traditional method.