Environmental Enrichment Improves the Recognition Memory in Adult Mice Following Social Isolation via Downregulation of Kv4.2 Potassium Channels.

Recognition memory is a cognitive process that enables us to distinguish familiar objects and situations from new items, which is essential for mammalian survival and adaptation to a changing environment. Social isolation (SI) has been implicated as a detrimental factor for recognition memory. The medial prefrontal cortex (mPFC) has been shown to carry information concerning the relative familiarity of individual stimuli, and modulating neuronal function in this region may contribute to recognition memory. The present study aimed to investigate the neuronal mechanisms in the mPFC of environmental enrichment (EE) on recognition memory in adult mice following SI. Mice were assigned into three groups: control, SI, and SI + EE groups. Novel location recognition (NLR) and novel object recognition (NOR) tests were performed to evaluate the recognition memory. The levels of Kv4 channels were assessed by qRT-PCR and western blotting. The effects of SI and SI + EE on the excitability of pyramidal neurons in the mPFC were measured using whole-cell recording. We found that SI led to a reduction in the excitability of pyramidal neurons. Specifically, we have identified that the reduction in the firing activity of pyramidal neurons resulted from alterations in the function and expression of Kv4.2 channels. Furthermore, EE regulated Kv4.2 channels, normalized the activity of pyramidal neurons, and restored the behavioral deficits following SI. Thus, the roles of Kv4.2 channels in excitability of pyramidal neurons suggest that the Kv4.2 channels present a promising therapeutic target for recognition memory impairment.

Broadly neutralizing antibodies derived from the earliest COVID-19 convalescents protect mice from SARS-CoV-2 variants challenge.

Coronavirus disease 2019 (COVID-19) was first reported three years ago, when a group of individuals were infected with the original SARS-CoV-2 strain, based on which vaccines were developed. Here, we develop six human monoclonal antibodies (mAbs) from two elite convalescents in Wuhan and show that these mAbs recognize diverse epitopes on the receptor binding domain (RBD) and can inhibit the infection of SARS-CoV-2 original strain and variants of concern (VOCs) to varying degrees, including Omicron strains XBB and XBB.1.5. Of these mAbs, the two most broadly and potently neutralizing mAbs (7B3 and 14B1) exhibit prophylactic activity against SARS-CoV-2 WT infection and therapeutic effects against SARS-CoV-2 Delta variant challenge in K18-hACE2 KI mice. Furthermore, post-exposure treatment with 7B3 protects mice from lethal Omicron variants infection. Cryo-EM analysis of the spike trimer complexed with 14B1 or 7B3 reveals that these two mAbs bind partially overlapped epitopes onto the RBD of the spike, and sterically disrupt the binding of human angiotensin-converting enzyme 2 (hACE2) to RBD. Our results suggest that mAbs with broadly neutralizing activity against different SARS-CoV-2 variants are present in COVID-19 convalescents infected by the ancestral SARS-CoV-2 strain, indicating that people can benefit from former infections or vaccines despite the extensive immune escape of SARS-CoV-2.

SAN-Net: Learning generalization to unseen sites for stroke lesion segmentation with self-adaptive normalization.

There are considerable interests in automatic stroke lesion segmentation on magnetic resonance (MR) images in the medical imaging field, as stroke is an important cerebrovascular disease. Although deep learning-based models have been proposed for this task, generalizing these models to unseen sites is difficult due to not only the large inter-site discrepancy among different scanners, imaging protocols, and populations, but also the variations in stroke lesion shape, size, and location. To tackle this issue, we introduce a self-adaptive normalization network, termed SAN-Net, to achieve adaptive generalization on unseen sites for stroke lesion segmentation. Motivated by traditional z-score normalization and dynamic network, we devise a masked adaptive instance normalization (MAIN) to minimize inter-site discrepancies, which standardizes input MR images from different sites into a site-unrelated style by dynamically learning affine parameters from the input; i.e., MAIN can affinely transform the intensity values. Then, we leverage a gradient reversal layer to force the U-net encoder to learn site-invariant representation with a site classifier, which further improves the model generalization in conjunction with MAIN. Finally, inspired by the "pseudosymmetry" of the human brain, we introduce a simple yet effective data augmentation technique, termed symmetry-inspired data augmentation (SIDA), that can be embedded within SAN-Net to double the sample size while halving memory consumption. Experimental results on the benchmark Anatomical Tracings of Lesions After Stroke (ATLAS) v1.2 dataset, which includes MR images from 9 different sites, demonstrate that under the "leave-one-site-out" setting, the proposed SAN-Net outperforms recently published methods in terms of quantitative metrics and qualitative comparisons.

Clinical features and genetic spectrum of Chinese patients with hereditary spastic paraplegia: A 14-year study.

Background: Hereditary spastic paraplegia (HSP) constitutes a group of clinically and genetically rare neurodegenerative diseases characterized by progressive corticospinal tract degeneration. The phenotypes and genotypes of HSP are still expanding. In this study, we aimed to analyse the differential diagnosis, clinical features, and genetic distributions of a Chinese HSP patients in a 14-year cohort and to improve our understanding of the disease. Methods: The clinical data of patients with a primary diagnosis of HSP at the initial visit to the Department of the Neurology, Peking University Third Hospital, from 2008 to 2022 were retrospectively collected. Next-generation sequencing gene panels (NGS) combined with a multiplex ligation-amplification assay (MLPA) were conducted. Epidemiological and clinical features and candidate variants in HSP-related genes were analyzed and summarized. Results: 54 cases (probands from 25 different pedigrees and 29 sporadic cases) from 95 patients with a primary diagnosis of HSP were finally confirmed to have a clinical diagnosis of HSP based on clinical criteria, including their clinical findings, family history and long-term follow-up. Earlier disease onset was associated with longer diagnostic delay and longer disease duration and was associated with a lower risk of loss of ability to walk independently. In addition, 20 candidate variants in reported HSP-related genes were identified in these clinically diagnosed HSP patients, including variants in SPAST, ALT1, WASHC5, SPG11, B4GALNT1, and REEP1. The genetic diagnostic rate in these 54 patients was 35.18%. Conclusion: Hereditary spastic paraplegia has high clinical and genetic heterogeneity and is prone to misdiagnosis. Long-term follow-up and genetic testing can partially assist in diagnosing HSP. Our study summarized the clinical features of Chinese HSP patients in a 14-year cohort, expanded the genotype spectrum, and improved our understanding of the disease.

A de novo c.113 T > C: p.L38R mutation of SPTLC1: case report of a girl with sporadic juvenile amyotrophic lateral sclerosis.

SPTLC1 has been implicated in hereditary sensory and autonomic neuropathy type 1 (HSAN1) and macular telangiectasia type2. Recent studies have reported mutations in SPLTC1 may cause juvenile amyotrophic lateral sclerosis (JALS), especially in the first transmembrane domain of SPTLC1(exon 2). In this study, we identified a novel heterozygous variant in exon 2, c.113 T > C: p. Leu38Arg, of SPTLC1 in a 12-year-old girl with sporadic JALS who experienced early-childhood-onset lower extremity spasticity followed by slowly progressive lower motor weakness and atrophy without sensory symptoms or signs. SPLTC1 is the first monogenic lipid metabolic disturbance that has been linked to ALS. The variant in exon 2 may impact on negative regulation of sphingolipid biosynthesis.

[Quercetin induces apoptosis of human breast cancer cells by activiting PTEN and inhibiting PI3K/AKT and JNK signaling pathways].

Objective To investigate the effect of quercetin on cell proliferation and apoptosis of MCF-7 human breast cancer cells, and effects of signaling pathways of PTEN/PI3K/AKT and c-Jun N-terminal kinase (JNK) signaling pathway. Methods MCF-7 cells were treated with quercetin (0, 20, 40, 60, 80, 100 µmol/L) CCK-8 assay was used to detect the cell proliferation, and cell apoptosis was assayed by flow cytometry. Hochesst33342 staining was used to observe the changes of nuclear number and karyotype, and flow cytometry was employed to detect cell apoptosis. The expression of PTEN and phosphorylated JNK (p-JNK) were detected by immunofluorescence cytochemistry, and the protein levels of PTEN, phosphorylated PI3K (p-PI3K), p-JNK and phosphorylated AKT (p-AKT) were detected by Western blot analysis. After treated with PI3K/AKT pathway inhibitor LY294002, the cell apoptosis and related protein expression were detected by the above methods again. Results With increased quercetin concentration, cell activity decreased and cell growth was inhibited in a concentration dependent manner; the nuclear concentration and apoptosis also increased. High concentration of quercetin significantly enhanced the expression and distribution of PTEN protein, decreased the levels of p-PI3K and p-AKT protein, and inhibited the expression of p-JNK protein. After adding LY294002 to inhibit PI3K/AKT, the apoptosis rate increased for cells treated with both LY294002 and quercetin; the expression of PTEN protein also showed an increase. Quercetin could significantly enhance the effect of LY294002 on p-PI3K/AKT/PTEN protein. Conclusion Quercetin can signeristically inhibit cell viability and induce cell apoptosis on MCF-7 cells, by up-regulating the expression of PTEN and down-regulating PI3K/AKT and JNK pathways.

Neuroimmune Crosstalk Between the Peripheral and the Central Immune System in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the degeneration and death of motor neurons. Systemic neuroinflammation contributes to the pathogenesis of ALS. The proinflammatory milieu depends on the continuous crosstalk between the peripheral immune system (PIS) and central immune system (CIS). Central nervous system (CNS) resident immune cells interact with the peripheral immune cells via immune substances. Dysfunctional CNS barriers, including the blood-brain barrier, and blood-spinal cord barrier, accelerate the inflammatory process, leading to a systemic self-destructive cycle. This review focuses on the crosstalk between PIS and CIS in ALS. Firstly, we briefly introduce the cellular compartments of CIS and PIS, respectively, and update some new understanding of changes specifically occurring in ALS. Then, we will review previous studies on the alterations of the CNS barriers, and discuss their crucial role in the crosstalk in ALS. Finally, we will review the moveable compartments of the crosstalk, including cytokines, chemokines, and peripheral immune cells which were found to infiltrate the CNS, highlighting the interaction between PIS and CIS. This review aims to provide new insights into pathogenic mechanisms and innovative therapeutic approaches for ALS.

An identical DCTN1 mutation in two Chinese siblings manifest as dHMN and ALS respectively: a case report.

Mutations in the DCTN1 gene have been found in patients with various neurodegenerative diseases, and the spectrum is still expanding. Here, we report a mutation in DCTN1 (c.175G > C, p.G59R) identified in two patients, who manifested dHMN and ALS, respectively, in an affected family. The clinical manifestations and eightyear follow-up suggested that this mutation is pathogenic. The phenomena observed in this family with the same DCTN1 mutation illustrate the clinical heterogeneity of DCTN1 gene mutations and expand our understanding of their genotype-phenotype relationships. Further research and functional experiments, especially mutation at amino acid position 59 of DCTN1, are required.

Validation of the pathogenic role of rare DNAJC7 variants in Chinese patients with amyotrophic lateral sclerosis.

DNAJC7 has recently been recognized as a novel amyotrophic lateral sclerosis (ALS) risk gene. To date, few studies have screened DNAJC7 mutations in Chinese population. Further studies are needed to clarify the clinical and genetic features of DNAJC7-related ALS. Sporadic ALS (sALS) patients and controls were enrolled in this study. Variants were detected by whole-exome sequencing and validated via Sanger sequencing. Gene-based burden analysis was conducted. Potentially damaging variants in DNAJC7 were identified in 3 sALS patients. The frequency of bulbar onset was significantly higher in DNAJC7-related ALS patients than in the whole group. However, burden analysis showed no enrichment of rare DNAJC7 variants in sALS patients. Reported variant N369T showed no significant difference in distribution among different groups. In conclusion, DNAJC7 variants may be associated with ALS but not play a main role in Chinese patients. DNAJC7-related ALS patients tended to have a bulbar onset. Our study supported the pathogenic role of DNAJC7 in ALS and expanded the phenotypic and genetic spectrum of DNAJC7-related ALS.

Activation of chicken macrophages during in vitro stimulation and expression of immune genes.

OBJECTIVE To characterize activation and expression of immune genes of chicken macrophages after in vitro stimulation with lipopolysaccharide (LPS) and mouse erythrocytes. ANIMALS Five 15-day-old chickens and 2 BALB/c mice. PROCEDURES Macrophages were extracted from chicken bone marrow or peripheral blood and then stimulated with cytokines secreted from cell lines L929 and HD11. Stimulated chicken macrophages were further cocultured with LPS or mouse erythrocytes, and gene transcription of some distinctive cytokines was detected by use of a real-time PCR assay. RESULTS Morphological features and phagocytic function of macrophages were characterized. Activated macrophages had an elongated shape with a large cell nucleus, and they had phagocytic function. Distinctive genes encoding the surface marker gene CD11b were identified; high quantities of CD11b were transcribed. Relative transcription of chicken genes BF and BL in mature cells cocultured with both stimuli was lower than for control cells. However, the quantity of genes encoding M1- or M2-distinctive cytokines (interleukin [IL]-1ß, IL-10, IL-12, inducible nitric oxide synthase, tumor necrosis factor-α, and transforming growth factor-ß) that were transcribed differed significantly between stimulation with LPS and mouse erythrocytes. CONCLUSIONS AND CLINICAL RELEVANCE Chicken macrophages were differentially stimulated by LPS and mouse erythrocytes, which suggested that in vitro stimulation can distinctly influence the transcription and expression of immune genes of chicken macrophages.

Grass carp (Ctenopharyngodon idellus) invariant chain of the MHC class II chaperone protein associates with the class I molecule.

The invariant chain (Ii) is an important immune molecule, as it assists major histocompatibility complex (MHC) class II molecules to present antigenic peptides. The relationship between the Ii and MHC molecules in teleosts remains poorly understood. This study focused on the molecular structure of grass carp Ii (gIi), its organ distribution, correlations with gene transcription, and the association with MHC. gIi cDNA was cloned using designed degenerate primers and the rapid amplification of cDNA ends method (RACE). The gIi sequence was 92%-96% similar to that of other teleosts, but only 52%-67% similar to that of mammals, respectively. The gIi gene was distributed in all 12 organs examined by PCR. The gIi gene transcription levels were markedly higher in organs enriched with immune cells than in other organs (P < 0.01). Moreover, positive correlations were detected between transcription levels of the gIi and gMhcI or II genes in different organs (r = 8.415-8.523, P = 0.001). The gIi co-localized on endomembrane systems with either class I or II molecules in co-transfected cells observed by a laser confocal. Further testing confirmed that the gIi bound gMHCI and II molecules. Taken together, these results indicate that the gIi is associated with MHC class I and II molecules, suggesting homology of both MHC molecules.

[Effect difference between two segments in invariant chain CLIP on humoral immune].

OBJECTIVE: To compare the effect between two segments (PBS and GBS) of Class II -associated invariant chain peptide (CLIP) of invariant chain (Ii) on humoral immune by immune carrier. METHODS: First six hybrids containing Newcastle disease virus (NDV) epitope F2 and Ii segments (Cyt/TM/F2, Cyt/TM/F2/GBS, Cyt/TM/PBS/F2, Cyt/TM/F2/TRIM, Cyt/TM/F2/GBS/TRIM, Cyt/TM/PBS/F2/TRIM) were reconstructed respectively. Then they were inserted into the prokaryotic expression vector pET-32a and transformed into E. coli Rosetta (DE3) to induce the expression of the recombinant proteins. Finally mice were immunized with these purified fusion proteins, the specific antibody titers were detected with ELISA, to compare and analyze the effect among different groups on the immune response. RESULTS: All the six groups immunized with these hybrids increased antibody titers (from 1.5-fold to 4.9-fold, respectively) compared with the group immunized with F2 alone. Within the above six groups, the hybrids containing either PBS or GBS had higher antibody titers from 1.6-fold to 2.4-fold than the hybrids without the both segments. However, the group of the hybrid containing PBS had a 1.5-fold antibody titer higher than the group of GBS hybrid. CONCLUSION: Ii cytosolic and transmembrane domains could increase the immune response, while the segment PBS behaved better than GBS in an immune vector based on Ii.

[Preparation and characterization of polyclonal antibody against Ia-associated invariant chain of Muscovy Duck (Cairina moschata)].

OBJECTIVE: To prepare polyclonal antibody against invariant chain of Muscovy duck (Cairina moschata) (MDIi) and identify its reaction with MDIi extracted from tissues of Muscovy duck. METHODS: MDIi was amplified by PCR and used to construct the prokaryotic expression vector of pET-32a/MDIi by linking with the plasmid of pET-32a. Then pET-32a/MDIi was transformed into E.coli Rosetta to induce the prokaryotic expression. After identified by SDS-PAGE, prokaryotic expression products were further purified from running gel of SDS-PAGE and injected into mice to prepare polyclonal antibody against MDIi. The titer and specificity of the polyclonal antibody against MDIi were analyzed by indirect ELISA and Western blotting, respectively. The intensity of reaction between the polyclonal antibody and MDIi extracted from tissues of Muscovy duck was also identified by indirect ELISA. RESULTS: The prokaryotic expression vector pET-32a/MDIi was successfully constructed. About 40 kD recombinant proteins of MDIi were confirmed to be expressed in the form of inclusion body in Rosetta. Polyclonal antibody against MDIi with a titer of 1:128 000 was obtained from the immunized mice and its high specificity was demonstrated by Western blotting. The titer of reaction between the polyclonal antibody and MDIi was 1:32 000. CONCLUSION: The polyclonal antibody against MDIi was successfully prepared with a high titer and specificity. It has a strong immune reaction with MDIi extracted from tissues of Muscovy duck.

Allele-dependent association of chicken MHC class I molecules with the invariant chain.

Invariant chain (Ii) associates with MHC class I molecules in cross-presentation pathway in mouse, but the association of Ii with MHC class I molecules in chicken was not clear. In this study we selected five typical alleles from about 100 B-FA and some B-FB sequences and tested. Confocal microscopy revealed that only two alleles of α chain (CD type) rather than ß chain showed incomplete co-localization with Ii own, or as a combined cytosolic and transmembrane domains in the co-transfected 293T cells, while other allele types, CK and CL, had no this ability. Co-immunoprecipitation (IP) indicated that one of both alleles bound only full-length Ii. Interestingly, further analysis of five sequences showed that the types CK had a tail and the most variable sites (39/44) localize within MHC I α1 and α2 regions, this suggests that the tail and multi-sites are crucial to the association with Ii. Additionally, qRT-PCR revealed that Ii transcription levels in different organs were positively correlated with those of B-FA or B-FB gene. These results suggest that the allele-dependent association of chicken MHC class I molecules with its Ii with bases on a multi-site determinative spatial structure and that Ii as carrier potentially perform similar roles in MHC II as well as in MHC I antigen peptide presentation.

Cross-species association of quail invariant chain with chicken and mouse MHC II molecules.

There are different degrees of similarity among vertebrate invariant chains (Ii). The aim of this study was to determine the relationship between quail and other vertebrate Ii MHC class II molecules. The two quail Ii isoforms (qIi-1, qIi-2) were cloned by RACE, and qRT-PCR analysis of different organs showed that their expression levels were positively correlated with MHC II gene (B-LB) transcription levels. Confocal microscopy indicated that quail full-length Ii co-localized with MHC II of quail, chicken or mouse in 293FT cells co-transfected with both genes. Immunoprecipitation and western blotting further indicated that these aggregates corresponded to polymers of Ii and MHC class II molecules. This cross-species molecular association of quail Ii with chicken and mouse MHC II suggests that Ii molecules have a high structural and functional similarity and may thereby be used as potential immune carriers across species.

Boosting immune response with the invariant chain segments via association with non-peptide binding region of major histocompatibility complex class II molecules.

BACKGROUND: Based on binding of invariant chain (Ii) to major histocompatibility complex (MHC) class II molecules to form complexes, Ii-segment hybrids, Ii-key structure linking an epitope, or Ii class II-associated invariant chain peptide (CLIP) replaced with an epitope were used to increase immune response. It is currently unknown whether the Ii-segment cytosolic and transmembrane domains bind to the MHC non-peptide binding region (PBR) and consequently influence immune response. To investigate the potential role of Ii-segments in the immune response via MHC II/peptide complexes, a few hybrids containing Ii-segments and a multiepitope (F306) from Newcastle disease virus fusion protein (F) were constructed, and their binding effects on MHC II molecules and specific antibody production were compared using confocal microscopy, immunoprecipitation, western blotting and animal experiments. RESULTS: One of the Ii-segment/F306 hybrids, containing ND (Asn-Asp) outside the F306 in the Ii-key structure (Ii-key/F306/ND), neither co-localized with MHC II molecules on plasma membrane nor bound to MHC II molecules to form complexes. However, stimulation of mice with the structure produced 4-fold higher antibody titers compared with F306 alone. The two other Ii-segment/F306 hybrids, in which the transmembrane and cytosolic domains of Ii were linked to this structure (Cyt/TM/Ii-key/F306/ND), partially co-localized on plasma membrane with MHC class II molecules and weakly bound MHC II molecules to form complexes. They induced mice to produce approximately 9-fold higher antibody titers compared with F306 alone. Furthermore, an Ii/F306 hybrid (F306 substituting CLIP) co-localized well with MHC II molecules on the membrane to form complexes, although it increased antibody titer about 3-fold relative to F306 alone. CONCLUSIONS: These results suggest that Ii-segments improve specific immune response by binding to the non-PBR on MHC class II molecules and enabling membrane co-localization with MHC II molecules, resulting in the formation of relatively stable MHC II/peptide complexes on the plasma membrane, and signal transduction.

[Cloning of pigeon invariant chain (Ii) gene by RACE].

In order to compare the structure and function of pigeon invariant chain (pIi) gene with other avian's, pIi gene was cloned using a method of RACE (Rapid Amplification of cDNA Ends). Firstly, according to high conservative nucleotide sequence of homologous fragment in avian invariant chain (Ii) gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from pigeon spleen cell RNA by PCR. Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of pIi gene were cloned by RACE respectively. Finally a complete cDNA of pIi was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that pIi gene is 1,050 bp in length (GenBank No. AY904337), which includes an open reading frame of 633 bp encoding a precursor protein with 211 amino acid residues. In comparison with the nucleotide sequences of other species' Ii genes, pIi is similar to chicken's, showing an overall identity of 82.8 with chicken and over 52.0 with human and other mammalian animals. In addition, some amino acid residues in Ii molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.

Molecular cloning and mRNA expression of duck invariant chain.

In the present study we identified a duck invariant chain (Ii) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1190 bp in length and contained a 669 bp open reading frame. An alternative transcript encoding a thyroglobulin (Tg)-containing form of Ii, named duck Ii-2, was also found in duck. The putative amino acid sequence of duck Ii-1 showed an 82% similarity to chicken Ii-1 and about 60% similarity to its mammalian homologues. The similarity of the Tg domain between duck and chicken Ii-2 was 96%, and about 70% between duck and mammalian Ii. The result of RT-PCR showed that Ii mRNA was extensively expressed in various tissues. High levels of both Ii-1 and Ii-2 mRNA were observed in the spleen and bursa of Fabricius. The predicted three-dimensional (3D) structures of duck Ii trimerization and Tg domain are similar to the corresponding regions of human Ii analyzed by comparative protein modeling. These findings indicate that the two isoforms of duck Ii, which strongly expressed in the major immune organs, share structural identity with human Ii.

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing.

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.