Characteristics of human large granular lymphocytes and relationship to natural killer and K cells

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcγR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcγR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Treatment with anti-TNF monoclonal antibody (c5N) reduces the extent of induced endometriosis in the baboon

BACKGROUND: Inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha), are important in the pathogenesis of endometriosis. We assessed the efficacy of anti-TNF monoclonal antibody (mAb, c5N), known to prevent induced endometriosis in baboons, in reducing established endometriosis in baboons. METHODS: This prospective, randomized, blinded, controlled study was conducted in baboons at the Institute of Primate Research (IPR), Nairobi, Kenya. Endometriosis was induced in 18 adult female baboons (Papio anubis) with regular menstrual cycles and a normal pelvis; the extent of endometriosis was documented by videolaparoscopy 25 days later. The baboons were then randomly assigned to receive a single infusion of either placebo (n=7, 5 ml/kg) or c5N (n=11, 5 mg/kg). Follow-up laparoscopy was performed 25 days later to document any differences in the number, surface area and estimated volume of lesions between the two groups and between the first and the second laparoscopies in each group. Representative biopsies of at least one endometriotic lesion per baboon were obtained at the final laparoscopy. RESULTS: Significant reductions in total surface area, estimated total volume of endometriotic lesions and both number and surface area of red lesions were observed after treatment with c5N, but not after placebo treatment, when compared to the initial laparoscopy. Conversely, a significant increase in the number of typical and red lesions was observed after placebo treatment when compared to the initial laparoscopy. Neither c5N nor placebo treatment affected the menstrual cycle. CONCLUSION: In baboons with induced endometriosis, anti-TNF-mAb (c5N) treatment significantly reduced the extent of endometriosis, mainly due to reducing both the number and surface area of red lesions. These findings suggest that anti-TNF-mAb therapy may have therapeutic potential for active peritoneal endometriosis.     

Unconventional T‐cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage

It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II–peptide complex termed type A and type B, and that the two conformers of the same peptide–MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T‐cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T‐cell recognized the same core epitope. The results are consistent with the generation of aggrecan‐specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan‐induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.     

Characterization of melanocyte stimulating hormone receptor variant alleles in twins with red hair

The association between MSHR coding region variation and hair colour in humans has been examined by genotyping 25 red haired and 62 non-red Caucasians, all of whom were 12 years of age and members of a twin pair study. Twelve amino acid substitutions were seen at 11 different sites, nine of these being newly described MSHR variants. The previously reported Val92Met allele shows no association with hair colour, but the three alleles Arg151Cys, Arg160Trp and Asp294His were associated with red hair and one Val60Leu variant was most frequent in fair/blonde and light brown hair colours. Variant MSHR genotypes are associated with lighter skin types and red hair (P < 0.001). However, comparison of the MSHR genotypes in dizygotic twin pairs discordant for red hair colour indicates that the MSHR gene cannot be solely responsible for the red hair phenotype, since five of 13 pairs tested had both haplotypes identical by state (with three of the five having both identical by descent). Rather, it is likely that additional modifier genes exist, making variance in the MSHR gene necessary but not always sufficient, for red hair production.      

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.      

Evaluation of maternal plasma creatine kinase activity as a marker of abnormal early pregnancy

We have tested the value of maternal plasma creatine kinase activity for diagnosing ectopic pregnancies obtained after in-vitro fertilization and embryo transfer. Plasma creatine kinase was assayed in 57 patients: 20 normal, 23 miscarriages and 14 ectopic pregnancies, for a total of 240 samples. All values were in the lower part of the normal range except only one in a miscarrying patient. A statistically significant difference was observed for a cut-off value of 45 IU/l between normal and ectopic pregnancies. However, for this cut-off point, the measurement of plasma creatine kinase activity had a sensitivity of 0.50 and a specificity of 0.76 for the diagnosis of ectopic pregnancy. The positive predictive value was 0.69. Creatine kinase activity measurements are thus of no practical value in this particular population, in which an early and specific marker of ectopic implantation would be of paramount interest. The association of human chorionic gonadotrophin (HCG) determinations and ultrasound scanning of the pelvis still remain the best paraclinical support for an early diagnosis of ectopic implantation.      

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.