Tissue-specific Compartmentalization Research Articles

P-547 Confined placental mosaicism for inherited segmental aneuploidies in miscarriages traces early karyotype self-correction events

Abstract Study question Does tissue-specific compartmentalization of segmental aneuploidies in first-trimester miscarriages reflect their postzygotic de novo origin or correction of inherited copy number variants (CNV)? Summary answer Most of mosaic CNVs (84%) in spontaneous abortions with normal karyotype arose de novo, but 16% resulted from postzygotic tissue-specific correction of maternally inherited variants. What is known already Chromosomal mosaicism is a hallmark of early human embryogenesis affecting significantly developmental potential of blastocysts, it’s implantation and delivery rates. Most of the currently available data are related to mosaic aneuploidies for a whole chromosome in IVF cycles, whereas little is known about origin and fate of mosaic segmental CNVs due to inherent limitations of NGS and aCGH for detection of copy number changes less for 5-10 Mb in size after single-cell whole genome amplification. Study design, size, duration Chorionic villi (CV) and extraembryonic mesoderm (EM) of miscarriages as well as maternal and paternal peripheral blood DNA samples were evaluated by aCGH+SNP arrays to detect the prevalence and tissue-specific distribution of segmental aneuploidies. As far as CV and EM are derivatives of trophectoderm and inner cell mass lineages, comparative analysis of both tissues allows to investigate the origin of mosaic CNVs by postzygotic de novo appearance or correction of the inherited variants. Participants/materials, setting, methods Eleven trios (mother, father, CV and EM samples from spontaneous abortion with normal karyotype by G-banding) were included in the study. Mean gestational age was 7.9 weeks. Mean maternal and paternal ages were 30.8 and 30.5 years, respectively. aCGH+SNP analysis was performed by SurePrint G3 Human CGH + SNP 4 × 180K microarrays (Agilent Technologies) with 13 kb median aCGH probes spacing. Main results and the role of chance Altogether 116 CNVs were detected in CV and EM samples from 11 abortions. Fifty four (46.6%) variants were present in both tissues, while 29 (25%) were confined by CV and 33 (28.4%) by EM. Most of tissue-specific segmental aneuploidies in CV (90%) and EM (79%) arose de novo, whereas others were inherited from parents. It is important that 5 maternally inherited microduplications in some clinically significant regions like 1p21.1 (103 kb in size), 2p21 (3.56 kb), 3p22.3 (128 kb), 7q36.3 (42.7 kb), and Xq28 (182 kb) were confined by EM, whereas two at 4p16.3 (22.3 kb) and 19q13.43 (2.2 kb) by CV. No cases of tissue-specific maternally inherited microdeletions or paternal CNVs were observed in our study. One can suggest that confined placental mosaicism for inherited maternal microduplications arisen by trisomy rescue mechanism with loss of additional maternal chromosome copy in CV or EM after its divergence around the time of blastocyst implantation. This hypothesis is confirmed by comparative SNP-haplotype analysis of DNA samples from both CV and EM tissues as well as parental samples in the subchromosomal regions of microduplications and adjusted loci. Limitations, reasons for caution Control group of induced (social) abortions of the same developmental terms are required to estimate the prevalence and distribution of mosaic segmental aneuploidies in embryonic and extraembrionic tissues during normal human embryo development. Wider implications of the findings Tracing of tissue-specific mosaic segmental aneuploidies in first-trimester miscarriages allows to estimate the level of karyotype instability and self-correction during human embryo development. This study was supported by the Russian Science Foundation, № 21-65-00017, https://rscf.ru/project/21-65-00017/ Trial registration number not applicable

D-Serine Production, Degradation, and Transport in ALS: Critical Role of Methodology

In mammalian systems, D-serine is perhaps the most biologically active D-amino acid described to date. D-serine is a coagonist at the NMDA-receptor, and receptor activation is dependent on D-serine binding. Because D-serine binding dramatically increases receptor affinity for glutamate, it can produce excitotoxicity without any change in glutamate per se. D-serine is twofold higher in the spinal cords of mSOD1 (G93A) ALS mice, and the deletion of serine racemase (SR), the enzyme that produces D-serine, results in an earlier onset of symptoms, but with a much slower rate of disease progression. Localization studies within the brain suggest that mSOD1 and subsequent glial activation could contribute to the alterations in SR and D-serine seen in ALS. By also degrading both D-serine and L-serine, SR appears to be a prime bidirectional regulator of free serine levels in vivo. Therefore, accurate and reproducible measurements of D-serine are critical to understanding its regulation by SR. Several methods for measuring D-serine have been employed, and significant issues related to validation and standardization remain unresolved. Further insights into the intracellular transport and tissue-specific compartmentalization of D-serine within the CNS will aid in the understanding of the role of D-serine in the pathogenesis of ALS.

Genetic and functional heterogeneity of CNS-derived tat alleles from patients with HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) demonstrates a high degree of viral diversity which has an impact on viral fitness. Genetic compartmentalization of HIV-1 proteins between central nervous system (CNS) and lymphoid tissues is well established and reflects altered requirements for HIV-1 replication in macrophages/microglia, brain-specific immune selection pressures and possibly the timing of virus invasion of the CNS. Tat-encoding mRNA has been detected in the CNS of HIV-1 infected individuals and its neurotoxic effects in the CNS are well documented. However, while CNS-derived tat sequences have demonstrated significant diversity, the effect of this molecular diversity on transcriptional regulation and its impact on the pathogenesis of HIV-associated dementia (HAD) remains unclear. In this study, we cloned and characterised 44 unique tat alleles from brain, cerebral spinal fluid, spinal cord and blood/lymphoid tissue-derived HIV-1 isolates from five subjects with HAD. While phylogenetic analyses revealed tissue-specific compartmentalization of Tat variants for two patients, broad compartmentalization across the panel of tissue-derived viruses was not observed. Despite the lack of consistent tissue-specific compartmentalization, sequence variations within patients segregated CNS and non-CNS tat alleles. These amino acid alterations predominated within the transactivation domain of Tat and could account for alterations in the ability of particular Tat proteins to transactivate the LTR. Although a subset of patients demonstrated reduced transactivation capacity among CNS-derived Tat proteins compared to those from matched lymphoid tissues, overall Tat proteins from the CNS to lymphoid compartments maintained similar levels of transactivation function. Together, these data suggest that despite the observed heterogeneity in tat alleles isolated from matched lymphoid to CNS compartments, Tat function is maintained, highlighting the importance of Tat function in HIV-1 neuropathogenesis.

Neuropathogenic SIVsmmFGb Genetic Diversity and Selection-Induced Tissue-Specific Compartmentalization During Chronic Infection and Temporal Evolution of Viral Genes in Lymphoid Tissues and Regions of the Central Nervous System

SIVsmmFGb is a lentivirus swarm that induces neuropathology in over 90% of infected pigtailed macaques and reliably models central nervous system HIV infection in people. We have previously studied SIVsmmFGb genetic diversity and compartmentalization during acute infection, but little is understood about diversity and intertissue compartmentalization during chronic infection. Tissue-specific pressure appeared to affect the diversity of Nef sequences between tissues, but changes to the Env V1 region and Int diversity were similar across all tissues. At 2 months postinfection, compartmentalization of the SIVsmmFGb env V1 region, nef, and int was noted between different brain regions and between brain regions and lymph nodes. Convergent evolution of the nef and env V1 region, and divergent evolution of int, was noted between compartments and all genes demonstrated intratissue temporal segregation. For the env V1 region and nef, temporal segregation was stronger in the brain regions than the periphery, but little difference between tissues was noted for int. Positive selection of the env V1 region appeared in most tissues at 2 months postinfection, whereas nef and int faced negative selection in all tissues. Positive selection of the env V1 region sequences increased in some brain regions over time. SIVsmmFGb nef and int sequences each saw increased negative selection in brain regions, and one lymph node, over the course of infection. Functional differences between tissue compartments decreased over time for int and env V1 region sequences, but increased for nef sequences.

Reduced genetic diversity in lymphoid and central nervous system tissues and selection-induced tissue-specific compartmentalization of neuropathogenic SIVsmmFGb during acute infection.

The simian lentivirus strain SIVsmmFGb is a viral swarm population inducing neuropathology in over 90% of infected pigtailed macaques and serves as a reliable model for HIV neuropathogenesis. However, little is understood about the genetic diversity of this virus, how said diversity influences the initial seeding of the central nervous system and lymph nodes, or whether the virus forms distinct genetic compartments between tissues during acute infection. In this study, we establish that our SIVsmmFGb stock virus contains four genetically distinct envelope V1 region groups, three distinct integrase groups, and two Nef groups. We demonstrate that initial central nervous system and lymph node seeding reduces envelope V1 and integrase genetic diversity but has a variable effect on Nef diversity. SIVsmmFGb envelope V1 region genes from the basal ganglia, cerebellum, and hippocampus form distinct genetic compartments from each other, the midfrontal cortex, and the lymph nodes. Basal ganglia, cerebellum, hippocampus, and midfrontal cortex-derived nef genes all form distinct genetic compartments from each other, as well as from the lymph nodes. We also find basal ganglia, hippocampus, and midfrontal cortex-derived integrase sequences forming distinct compartments from both of the lymph nodes and that the hippocampus and midfrontal cortex form separate compartments from the cerebellum, while the axillary and mesenteric lymph nodes compartmentalize separately from each other. Compartmentalization of the envelope V1 genes resulted from positive selection, and compartmentalization of the nef and integrase genes from negative selection. These results indicate restrictions on virus genetic diversity during initial tissue seeding in neuropathogenic SIV infection.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis–encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis.

Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis. The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate. We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues-cytotrophoblast and extraembryonic mesoderm-of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture. An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes. The overall rate of numerical chromosomal abnormalities in these cells was 53%. Both typical and rare forms of karyotype imbalance were found. The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding. Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast. The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development. The effect of aneuploidy on selection may differ, however, depending on the specific chromosome. The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; P<0.001). Confined placental mosaicism was found in 25% of the embryos. The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated.

Genetic and functional analysis of full-length human immunodeficiency virus type 1 env genes derived from brain and blood of patients with AIDS.

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.

Tissue-Specific Placental Mosaicism for Autosomal Trisomies in Human Spontaneous Abortuses: Mechanisms of Formation and Phenotypical Effects

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.

Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern.

Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.

In contrast to sessile cell types, lymphocytes migrate through the vasculature to become diffusely distributed in tissues or organized in lymphoid structures. A complex array of adhesion molecules including selectins, integrins and their counter-receptors mediate lymphocyte homing and migration into tissues and may be constitutively expressed or induced. However, the molecules that mediate the tissue-specific retention of lymphocytes within the parenchyma have not been identified. Along the epithelium at the basolateral surface of enterocytes, intestinal intraepithelial lymphocytes are found. These T cells of the mucosal immune system serve as a model for the tissue-specific compartmentalization of lymphocytes. We investigated whether the localization of these intestinal intraepithelial lymphocytes could be mediated by specific interactions between adhesion molecules expressed selectively on this subpopulation of T cells and tissue-restricted adhesion molecules on epithelial cells. Here we show that heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro are mediated by E-cadherin and the alpha E beta 7 integrin.