Distinct organization of adaptive immunity in the long-lived rodent Spalax galili.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

High-quality full-length immunoglobulin profiling with unique molecular barcoding.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

[The oligoclonal expansion of T cells: study of its stability over time].

A novel experimental approach to the investigation of the repertoire of peripheral T lymphocytes of patients suffering from ankylosing spondylitis (AS) is proposed. This approach is based on the wide-range sequencing of cDNA of the beta-chain of the T-cellular receptor (TcR). The results of the analysis of the diversity of sequences of the TcR antigen-binding domain (CDR3) inside the total pool of one patient with AS are presented by the example of the second V family (BV2) of TcR. The expansion of six independent TcR-expressing clones of T cells with a similar amino acid sequence of the CDR3 domains was proposed based on the results of the comparative structural analysis of the clone libraries of the cDNA of TcR BV2. The long-time stable expansion of these T clones was demonstrated during the development of the disease by specific monitoring.

Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

[A family of genes of multidomain free lectins from a planarian: structure, expression, and use as markers for regeneration monitoring ]. / Semeistvo genov multidomennykh svobodnykh lektinov planarii: struktura, ékspressiia i ispol'zovanie v kachestve markerov dlia nabliudeniia za regeneratsiei.

A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Cloning and analysis of a new neurogene in the mouse]. / Klonirovanie i analiz novogo neirogena myshi.

The previously described gene sip1 belongs to transcription factors of the zinc finger family. It has been ascertained recently that this gene is involved in TGF signaling cascade. Mutations in human gene sip1 cause Hirschprung syndrome. The expression of gene sip1 during embryonic mouse development was studied by in situ hybridization and immunostaining. Starting at E12.5, sip1 transcripts are present in a number of tissues: in the cortical plate, ventricular zone of the basal ganglion, thalamus, pons and midbrain, in specific nuclei of the brain stem and in the dorsal part of the spinal cord. In the developing cerebral cortex, sip1 expression is region-specific. In the brain of adult mice, sip1 expression is mostly detected in hippocampus, dentate gyrus, and white matter of the neocortex. Sip1 protein expression in the cerebral cortex is mostly confined to glutamatergic neurons.

Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.