Quantifying mesenchymal stem cells in the mononuclear cell fraction of bone marrow samples obtained for cell therapy.

AIMS: The use of bone marrow mononuclear cells (BMMNCs) as a source of mesenchymal stem cells (MSCs) for therapy has recently attracted the attention of researchers because BMMNCs can be easily obtained and do not require in vitro expansion before their use. This study was designed to quantify the MSC population in bone marrow (BM) samples obtained for cell therapy using flow cytometry to detect the CD271 antigen. MATERIAL AND METHODS: Autologous BM was obtained by posterior superior iliac crest aspiration under topical anesthesia. Mononuclear cells isolated from the BM aspirate on a Ficoll density gradient were used to treat patients with pressure ulcer (n = 13) bone nonunions (n = 3) or diabetic foot ulcers (n = 5). RESULTS: Our flow cytometry data revealed a low percentage as well as a high variability among patients of CD271(+)CD45(-) cells (range, 0.0017 to 0.0201%). All cultured MSC adhered to plastic dishes showing a capacity to differentiate into adipogenic and osteogenic lineages. CONCLUSIONS: Our findings suggested that the success of cell therapy was independent of the number of MSCs present in the BM aspirate used for autologous cell therapy.

HLA-B27 polymorphism at position 116 critically influences the association with TAP/tapasin, intracellular trafficking and conformational homodimers formation.

HLA-B27 confers susceptibility to ankylosing spondylitis but AS disease mechanisms remain unknown. We determined here the effect of polymorphism and tapasin dependence on the expression, intracellular maturation and homodimer formation among HLA-B27 subtypes. We found that B*2709 with a histidine at position 116 was strongly associated with the transporter associated with antigen processing complex, correlated with lower, non-conformational expression on the cell surface, delayed maturation rate and minimal conformational and non-conformational homodimer formation. In contrast, B*2705 showed a low dependence for transporter associated with antigen processing, faster intracellular maturation and increased levels of homodimeric forms. The absence of tapasin significantly influenced the rate of intracellular maturation of B*2709, showing faster transport out of the endoplasmic reticulum, but similar to that of B*2705. All B27 subtypes examined were unable to express conformational homodimeric forms in the absence of tapasin. This study suggests that HLA-B27 polymorphism drives the tapasin dependency, rates of intracellular maturation and expressions of homodimers.

The relationship of anti-MICA antibodies and MICA expression with heart allograft rejection.

The role of MICA antibodies in acute heart allograft rejection was examined utilizing 190 pre- and post-transplant serum samples from 44 patients collected during the first year after transplantation. MICA antibodies were detected by CDC test on recombinant cell lines and by the newly developed Luminex MICA antibody detection assay. Additionally, MICA expression was analyzed by 'real time' RT-PCR and by immunohistochemistry in 10 endomyocardial biopsies. Only two subjects had HLA antibodies post-transplant. Nevertheless, MICA antibodies were found in a significant number of subjects. The prevalence of MICA antibodies was significantly higher among those with severe acute rejection (AR) than in those without rejection (60.7% vs. 14.3%, p = 0.0038 by CDC; 55.5% vs. 5.7%, p = 0.0020 by Luminex). In most cases, the appearance of MICA antibodies post-transplant precedes AR. Following transplantation, MICA up-regulation correlated with histological evidence of severe rejection. Monitoring for MICA antibodies post-transplant may be useful to establish new risk factors for acute rejection.

Expression of the neurotrophin receptor TrkB in the mouse liver.

Neurotrophins acting through Trk signal-transducing receptors play essential roles in the nervous system, and probably in some non-neuronal tissues. In the present study, we used RT-PCR, Western-blot and immunohistochemistry to investigate the occurrence and cellular localization of TrkB in the mouse liver, from newborns to 6 months. Furthermore, the structure of the liver in mice carrying a mutation in the trkB gene, resulting in a non-functional protein, was studied. The analysis of the DNA sequence showed that hepatic trkB gene is identical to the cerebral one, and TrkB mRNA and TrkB full-length protein (145 kDa) were detected at all the ages sampled. Immunohistochemistry revealed age-dependent changes in the pattern of TrkB expression. From 0 to 15 days, the TrkB was detected in morphologically and immunohistochemically identified monocyte-macrophage-dendric cells scattered throughout the organ, while in animals 3- and 6-months-old it was restricted to nerve fibres. Interestingly, there was a parallelism between TrkB expression by monocyte-macrophage-dendric cells and the presence of hepatic erythroblastic islands. In agreement with a possible role of TrkB on hepatic haematopoiesis, the liver of 15 days old TrkB (-/-) mice still contained erythroblastic islands, whereas they were absent in the wild-type littermates. Another striking finding was the absence of nerve profiles in the TrkB (-/-) animals. All together, present results support the role of TrkB in the murine liver in maintaining the innervation of the organ, and more importantly throughout an unknown mechanism in controlling the hepatic haematopoietic function.

The amino acid at position 97 is involved in folding and surface expression of HLA-B27.

HLA-B27 confers susceptibility to ankylosing spondylitis (AS) but the mechanism linking this association remains unknown. Other properties unrelated to its natural role of antigen presenting function may be important in disease pathogenesis. We determined here the impact of N97D substitution on the folding and expression of HLA-B*2704 transfected in the 721.221 cell line. The mutation at position 97 abolishes the surface expression of non-conformational (HC10) and conformational (ME1) forms. The expression of ME1 forms was found to be absent in B*2704 N97D by immunoprecipitation and flow cytometry of fixed and permeabilized cell experiments with the conformation-sensitive ME1 antibody. However, immunoblotting cell lysates with HC10 revealed the presence of unfolded heavy chain (HC) and HC-dimer forms. The impact of the N97D mutation in the exit from the endoplasmic reticulum (ER) was analysed by western blot after endoglycosidase-H treatment, and it was found that B*2704 N97D was retained and accumulated as unfolded molecules. We tested for mutant association with transporter associated with antigen processing (TAP), calnexin (CNX), calreticulin (CLR) and beta2 microglobulin (beta2m). The wild-type B*2704 and N97D mutants were associated with TAP, CNX and CLR, although HC10 forms of mutant N97D interact more weakly with TAP. Only folded molecules of HLA-B*2704 were associated with beta2m. Surprisingly, the peptide-binding assay demonstrated the ability of unfolded N97D molecules to bind high-affinity peptides. It has been suggested that AS may arise because of aberrant folding of HLA-B27 molecules within the ER. Future work must therefore aim to clarify the functional connection between the unfolded protein response pathway in response to the accumulation of HLA-B27 in the ER. This mutant could be useful as a model for the misfolding of HLA-B27.

Interaction between KIR3DL1 and HLA-B*57 supertype alleles influences the progression of HIV-1 infection in a Zambian population.

KIR and HLA loci are both highly polymorphic, and some HLA class 1 products bind and trigger cell-surface receptors specified by KIR genes. We examined whether KIR genes act in concert with HLA-B locus to control HIV-1 infection in a sample of Zambian patients. DNA samples from 88 Zambian patients with HIV-1 were examined. Patients were classified as either slow progressors (SP; n = 54) or rapid progressors (RP; n = 34) to AIDS. All were typed for HLA-B and KIR genes. Our results reveal an association between B*57 supertype (B*57s, which includes B*57 and B*58 alleles) and delayed progression to AIDS (p = 0.0007 by pc = 0.015; OR = 5.25). We also observed an increase incidence of Bw4-I80 in patients with slow progression (p = 0.001 by pc = 0.003, OR = 5). This increase was found to be secondary to B*57s. The presence of both KIR3DL1 and B*57S has a significant effect on progression to AIDS (p = 0.0008; OR = 5.61). B*57s genotypes with another HLA-B allele different from those in the trans position, which also had a specificity different to Bw4-I80 (Bw4-T80 or Bw6), was also greater in the SP than in the RP group (p = 0.00003; OR = 10.11). The presence of the inhibitory allele KIR3DL1 in combination with the HLA-B*57s alleles that contain the Bw4-I80 epitope, has a highly protective effect against progression to AIDS in Zambian patients.

The HLA-B*5703 allele confers susceptibility to the development of spondylarthropathies in Zambian human immunodeficiency virus-infected patients with slow progression to acquired immunodeficiency syndrome.

OBJECTIVE: To analyze the HLA distribution in a population of individuals from Zambia in order to establish a possible relationship between the progression of human immunodeficiency virus (HIV) infection and the development of spondylarthropathy (SpA). METHODS: A large epidemiologic analysis of rheumatology patients living in Zambia was performed in order to identify those who had SpA. We selected 64 patients with SpA and found that 54 also had HIV type 1 (HIV-1) infection; only 10 were HIV negative. Additionally, we selected 57 HIV-infected individuals without SpA and 43 healthy controls. Among all of the HIV-1-infected patients, 25 SpA-positive and 24 SpA-negative patients were classified as slow progressors to acquired immunodeficiency syndrome (AIDS), and 8 SpA-positive and 26 SpA-negative patients were classified as rapid progressors. All patients were typed for HLA-B alleles. RESULTS: Of the 64 patients with SpA, HIV infection was observed in 54 (84%). The frequency of B*5703 was increased in patients who were SpA positive and HIV positive compared with patients who were SpA negative and HIV positive (P = 0.0002, odds ratio [OR] 8.28). Among patients who were slow progressors to AIDS, this allele was overrepresented in those with SpA compared with those without SpA (corrected P = 0.001, OR 26.25). CONCLUSION: HLA-B*5703 seems to be a protective allele against the progression of HIV infection but could influence the increased incidence of SpA observed in this population.

TrkB mRNA and protein in mouse spleen: structure of the spleen of functionally deficient TrkB mice.

Whereas it is nowadays clear that neurotrophins are involved in the regulation of various aspects of the functioning of immune system, knowledge of their actual immunomodulatory roles is still fragmentary and incomplete. In this respect, knock-out mouse models remain particularly unexplored. In the present study, the expression of the TrkB neurotrophin receptor in murine spleen was addressed at the mRNA (reverse transcription/polymerase chain reaction) and protein (Western blot) levels. Once the presence of TrkB at both levels was demonstrated, the age-dependent changes in the pattern of expression of the receptor were analyzed and quantified, and TrkB-positive cells were identified by immunohistochemistry. TrkB-immunoreactive cells, identified as red pulp macrophages, were detected in the spleen throughout postnatal development and adult life; their numbers peaked at the age of 15 days. The absence of functional TrkB did not appear to result in morphological changes as assessed by light and electron microscopy of spleens from 15-day-old mice knockout for the trkB gene. The present results support the idea that, in the murine spleen, TrkB and its ligands are involved in macrophage physiology in a developmentally regulated fashion, but they do not seem to be essential for macrophage survival.

Psoriasis vulgaris and psoriatic arthritis share a 100 kb susceptibility region telomeric to HLA-C.

OBJECTIVE: To map the locus for susceptibility to psoriasis in patients with psoriatic arthritis. METHODS: Seventy-four patients with psoriatic arthritis and 95 patients with psoriasis vulgaris were included in this study. Polymorphic genes and microsatellite markers centromeric (C1_2_5) and telomeric (C1_4_4, OTF3, HCR and the corneodesmosin gene) to HLA-C were studied in an association analysis. Typing was also performed on a control population of 104 matched donors. RESULTS: The allele Cw*0602 was associated both with psoriasis [49 vs 21%, Pc<0.0003; odds ratio (OR)=3.6, aetiological factor (AF)=0.72] and with psoriatic arthritis (62 vs 21%, Pc<0.000001; OR=6.1, AF=0.83). In psoriatic patients a susceptibility region telomeric to HLA-C that includes C1_4_4 (56 vs 22%, Pc<0.0002; OR=4.39, AF=0.77), OTF3 (85 vs 60%, Pc<0.0002; OR=3.7, AF=0.73) and HCR (63 vs 26%, Pc<0.00001; OR=3.8, AF=0.74) was observed. In psoriatic arthritis patients the susceptibility region was delimited by HLA-C and C1_4_4 (384 allele, 56 vs 22%, Pc<0.0002; OR=4.37, AF=0.77). CONCLUSIONS: Comparing the susceptibility regions associated with the two diseases, an overlapping interval of 100 kb between HLA-C and OTF3, which might contain the psoriasis gene, can be defined.

Massive lymphocyte apoptosis in the thymus of functionally deficient TrkB mice.

The occurrence of TrkB in the murine thymus (15-day and 3-month old) was investigated by Northern blot, Western blot and immunohistochemistry. Furthermore, the thymus of 15-day-old mice carrying a non-functional mutation on trkB was analyzed. Both trkB mRNA and 145 kDa TrkB protein were detected. In addition, isolated lymphocytes and stromal cells also expressed this protein. The thymus of homozygous functionally TrkB-deficient animals showed structural and ultrastructural changes consistent with massive death of cortical lymphocytes, confirmed with TUNEL. Present results suggest a role for TrkB in maintaining the survival or preventing massive death of lymphocytes in the mammalian thymus.

Genetic variability, molecular evolution, and geographic diversity of HLA-B27.

HLA-B27 represents a family of 23 closely related alleles (B*2701-23) that differ at 24 amino acid positions. The pattern of polymorphisms of B27 was studied, with special reference to synonymous (Ks) and nonsynonymous (Ka) divergence among alleles. B27 alleles are characterized by the enhanced rate of nonsynonymous nucleotide substitution in the peptide-binding region (PBR). The percentage of substitutions between each of the B27 pairs ranges from 0.2%-3% in exons 2-3 to 1.8%-20.1% in the PBR. A phylogenetic analysis of all B27 alleles is described in order to identify subtypes with a common evolutionary history. These results, together with the phylogenetic trees obtained from the comparison between exons 2-3 and PBR indicate that polymorphism of B27 is selectively maintained. Most of the differences are clustered in the C/F pocket affecting the specific binding of antigenic peptides. Gene conversion and point mutation are the most important mechanisms responsible for B27 diversification. The interaction of selection, genetic drift, and recombination events is important for generating polymorphism at B27 alleles. We analyzed a large extended B27 positive population from different parts of the world. Our results indicate that B27 subtypes differ in their ethnic distribution, which may be the result of different genetic and geographical origins. Different factors such as genetic drift, bottleneck effect, and admixture among populations could contribute to the genetic constitution of B27. The striking correlation between the structural features of B27 and the ethnic distribution of these subtypes suggests a model of strong directional evolution, in which the subtypes could have arisen from B*2705.

Polymorphism in MICA rather than HLA-B/C genes is associated with psoriatic arthritis in the Jewish population.

The aim of this study was to examine whether the association of psoriatic arthritis (PsA) with human leukocyte antigen (HLA) class I genes is secondary to linkage disequilibrium with a nearby gene. We examined a sample of the Jewish population to investigate whether HLA-B/C and DR polymorphism is associated with susceptibility, or whether other closely related class I loci, such as the major histocompatibility complex class I chain-related gene A (MICA) and tumor necrosis factor (TNF), might play a role in disease development. Comparisons of different populations with different HLA profiles would be of value in identifying the candidate genes involved in PSA. Fifty-two patients with PsA and 73 random matched controls from a Jewish population were selected and DNA typed by polymerase chain reaction-single-strand oligonucleotide probe (PCR-SSOP) (HLA-C), PCR sequence-specific primers (PCR-SSP) (HLA-B, -DR), radioactive PCR (MICA-TM polymorphism in the transmembrane region), and PCR-RFLP (TNF). Some findings can be concluded from the study: (1) the frequency of HLA-B*5701, B*3801, B*39, B*27, Cw*0602, Cw*07, DRB1*0402, and DRB1*0701 were not found to be significantly increased in PsA; (2) no significant differences of TNFalpha promoter alleles at positions -308 and -238 were found between PsA and healthy controls; (3) the trinucleotide repeat polymorphism MICA-A9 was present at a higher frequency in PsA patients, (p(c) < 0.009, RR = 3.34, EF = 0.39); and (4) MICA-A9 polymorphism was found in linkage disequilibrium with HLA-B alleles (B*5701, B*3801) described to be associated with PsA in Caucasians. These results suggest that the MICA gene or other nearby gene(s) may be involved in the development of PsA, and it would thus appear that psoriasis vulgaris (PsV) and PsA are associated with different MHC susceptibility genes.

New insights regarding HLA-B27 diversity in the Asian population.

A polymerase chain reaction-sequence-specific primer (PCR-SSP) method which distinguishes all B27 alleles described at present (B*2701-23) has been developed. The distribution of B27 alleles was characterised in six different Asian populations. HLA-B*2705, 02, 04, 07, 22 (formerly B*2706) subtypes found in Asian populations differ in their ethnic distribution, which may be the result of different genetic and geographic origins. Furthermore, two novel B27 alleles were found in this study. B*2714 was identified in two Siberians, one of whom was a patient with ankylosing spondylitis. B*2715 was found in two patients with ankylosing spondylitis in Thais. These associations have not previously been reported in either ethnic group.

The OTF3 gene polymorphism confers susceptibility to psoriasis independent of the association of HLA-Cw*0602.

Psoriasis has been strongly associated to HLA-Cw6, but it remains unclear whether Cw6 itself or a closely linked gene is associated with the disease. The aim of this study was to clarify whether the HLA-C itself determines disease susceptibility or whether it acts only as a marker for the susceptibility allele. We examined a sample of 95 type I psoriasis patients and 104 Spanish matched controls to investigate whether HLA-Cw*0602 or other closely related class I loci, such as HLA-B and MICA (which are centromeric to HLA-C), or corneodesmosin gene and octamer transcription factor-3 genes (which are telomeric to HLA-C), might play a part in disease development. DNA samples were genotyped by polymerase chain reaction/sequence-specific primers (HLA-C), polymerase chain reaction/sequence-specific primers (HLA-B), radioactive polymerase chain reaction (MICA-TM polymorphism in the transmembrane region), and polymerase chain reaction/restriction fragment length polymorphism (protein S and octamer transcription factor-3). Our results show a significant increase of Cw*0602 in psoriasis patients (odds ratio = 3.64; pc < 0.0006). A significant association between the beta allele of octamer transcription factor-3 (HindIII) and psoriasis was also detected (odds ratio = 3.76; pc < 0.0003). The allele octamer transcription factor-3B (etiologic fraction = 0.62) was found to be more strongly associated to psoriasis vulgaris than Cw*0602 (etiologic fraction = 0.35) and the increase of octamer transcription factor-3 B allele is independent of the linkage disequilibrium with Cw*0602 as this was also found in Cw*0602 negative patients (odds ratio = 3.63; pc < 0.015, etiologic fraction = 0.55). We did not detect an association between the corneodesmosin gene and psoriasis. This fact suggests that the psoriasis susceptibility gene is located within a critical region of 147 kb, telomeric to HLA-C and centromeric to the corneodesmosin gene, and the association of Cw6 to psoriasis may be secondary to linkage disequilibrium.