Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection.

After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRalphabeta+CD8alphaalpha+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRalphabeta+CD8alphabeta+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.

Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo.

Tumor necrosis factor-alpha (TNFalpha) exists in two biologically active forms, a 26-kDa transmembrane form and a proteolytically cleaved and secreted form. We sequentially inactivated all three known cleavage sites of mouse TNFalpha by mutating the corresponding DNA sequences. A murine T cell hybridoma transfected with the nonsecretable mutant TNFalpha efficiently lysed L929 target cells in a cell contact-dependent manner and induced expression of vascular cell adhesion molecule-1 on mouse endothelioma cells. A genomic mouse TNFalpha clone encoding this mutant was subsequently introduced as a transgene into TNFalpha(-/-) lymphotoxin-alpha(-/-) mice. The 3' AU-rich regulatory elements of the TNF locus were maintained in the transgene to assure adequate gene regulation. Transmembrane TNFalpha transgenic mice were fully protected from endotoxic shock, and no TNFalpha bioactivity was detectable in the serum after stimulation with lipopolysaccharide. Activated CD4 T cells from these animals, however, lysed L929 cells in a cell contact-dependent way. After administration of lipopolysaccharide, transmembrane TNFalpha transgenic mice produced significantly higher levels of interleukin-12 than wild-type mice or TNF-deficient mice. This indicates that transmembrane TNFalpha may greatly affect the course of a cellular immune responses in vivo and exerts quantitatively and qualitatively distinct functions from secreted TNFalpha in vitro and in vivo.

CD5- CD8 alpha beta intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5- and CD5+ CD8 alpha beta IEL do not represent separate T cell lineages.

We followed alpha beta T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted alpha beta TCR-transgenic (tg) mice. The proportion of tg alpha beta TCR+ CD8 alpha beta IEL is reduced compared with CD8+ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8 alpha beta IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR+ CD5- CD8 alpha beta IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR+ CD5int or, even more pronounced, tg TCR+ CD5hi CD8 alpha beta IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5- CD8 alpha beta IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-alpha IEL monoclonal antibody 2E7. This clearly demonstrates that CD5-, and CD5+ CD8 alpha beta IEL cannot be considered as separate T cell lineages.

Normal binding of calcium to five fibrinogen variants with mutations in the carboxy terminal part of the gamma-chain.

Calcium ions are known to accelerate polymerization of fibrin monomers. Each of the two carboxy terminal domains of normal fibrinogen contains one high-affinity calcium binding site that seems to be situated close to the polymerization site in the gamma-chain. Most hitherto described functionally defective fibrinogen variants showed impaired clot formation. Since the tightly bound calcium ions may influence the conformation of the polymerization site, the question arises whether the abnormal clotting of a dysfibrinogen might be due to defective calcium binding. We investigated binding of calcium to fibrinogen and the effect of calcium on the clotting properties of five heterozygous fibrinogen variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinogens has one single amino acid substitution in the carboxy-terminal part of the gamma-chain: fibrinogen Claro (gamma 275 Arg-->His), Milano V (gamma 275 Arg-->Cys), Milano I (gamma 330 Asp-->Val), Bern I (gamma 337 Asn-->Lys), and Milano VII (gamma 358 Ser-->Cys). The shortest thrombin clotting time and the earliest onset of turbidity increase were observed in fibrinogen gamma 358 Ser-->Cys; both parameters were little affected by calcium concentration. In the variant gamma 337 Asn-->Lys, the thrombin time was abnormally prolonged at 0.01 mM Ca2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen gamma 330 Asp-->Val was barely improved at increasing calcium concentrations. Both variants with the substitution of gamma 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and the lowest turbidity of fibrin clots at any Ca2+ concentration used. High affinity calcium binding was found to be normal in all five fibrinogen variants studied, suggesting that their abnormal clotting was not due to defective binding of calcium. The gamma-chain in the fragment D1 derived from the variant gamma 337 Asn-->Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas fragments D1 from the other four gamma-chain variants as well as from normal fibrinogen were protected against plasmic degradation in the presence of 1 mM Ca2+.

Binding of calcium ions and their effect on clotting of fibrinogen Milano III, a variant with truncated A alpha-chains.

Calcium ions are known to be required for normal polymerisation of fibrin monomers. Normal human fibrinogen has three high-affinity calcium binding sites. Two of these are located in the D-domains whereas the third binding site was tentatively assigned either to the E-domain or to the C-terminal part of the A alpha-chain. Furthermore, binding of calcium to the low-affinity binding sites (n > or = 10) facilitates fibrin monomer polymerisation. In several abnormally clotting fibrinogen variants, the polymerisation defect was partially normalised following addition of calcium ions. In this study, we show normal binding of calcium to fibrinogen Milano III, a homozygous fibrinogen variant with truncated A alpha-chains (A alpha 452 Gly-Pro-Asp-->Trp-Ser-Stop). These results confirm that the C-terminal parts of the A alpha-chains beyond residue 451 Ile are not involved in calcium binding. The thrombin time was severely prolonged and the final clot turbidity was strongly reduced in fibrinogen Milano III. Moreover, calcium ions did not significantly improve the abnormal clotting behavior of this dysfibrinogen. The polymerisation defect in fibrinogen Milano III appears to be due to truncated A alpha-chains as well as to the disulphide-linked albumin.

A frameshift mutation in Exon V of the A alpha-chain gene leading to truncated A alpha-chains in the homozygous dysfibrinogen Milano III.

An inherited dysfunctional fibrinogen variant, denoted as fibrinogen Milano III, was found in a 13-year-old girl suffering from recurrent venous thrombosis. Plasma of the patient exhibited prolonged thrombin time and Reptilase time. Polymerization of fibrin monomers in the presence and absence of calcium ions was strongly impaired. SDS-polyacrylamide gel electrophoresis of reduced fibrinogen showed normal B beta- and gamma-chains, whereas no normal A alpha-chain was detected in the proposita. Immunoblot analysis with the monoclonal antibody Y18, detecting an epitope within the stretch of amino acids A alpha 1-51, revealed an A alpha-chain of about 50 kDa with an intact amino terminus. Immunoblotting with antibodies directed against serum albumin demonstrated the presence of albumin covalently linked to fibrinogen via a disulfide bridge. The structural defect of fibrinogen Milano III was determined by sequence analysis of a single-stranded fragment of genomic DNA amplified by polymerase chain reaction. An insertion of a thymine in the exon V of the A alpha-chain gene after the triplet ATT coding for IleA alpha 451 altered the reading frame and caused premature termination of the protein synthesis (Trp452(TGG)-Ser453(TCC)-Stop454(TGA)). In both parents, normal and mutant alleles were established, leading to duplication of the sequence pattern after the thymine insertion site, whereas the proposita is homozygous for the new mutation in the fibrinogen A alpha-chain gene.

A new substitution, gamma 358 Ser-->Cys, in fibrinogen Milano VII causes defective fibrin polymerization.

Fibrinogen Milano VII is a hereditary fibrinogen variant detected in a woman with no clinical symptoms of bleeding or thrombosis. Thrombin and reptilase clotting times were prolonged in six family members from three generations. Release of fibrinopeptides A and B was normal. Fibrin polymerization was strongly delayed both in the presence and in the absence of calcium. The structural defect was determined by sequence analysis of a 290-bp fragment of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet TCT coding for the amino acid residue gamma 358 was found to be replaced by TGT, resulting in the substitution gamma 358 Ser-->Cys. Immunoblot analysis demonstrated the presence of covalently linked fibrinogen albumin and fibrinogen (albumin)2 complexes. Albumin was released from fibrinogen Milano VII by limited reduction with 2-mercaptoethanol. Fibrin polymerization was not normalized after removal of albumin from fibrinogen Milano VII, suggesting that the delayed clot formation is not due to steric hindrance caused by bound albumin but by substitution of gamma 358 Ser by Cys itself. Our results indicate that the residue gamma 358 Ser is essential for normal expression of the carboxy terminal polymerization site on the fibrinogen gamma-chain.

Fibrinogen Milano V: a congenital dysfibrinogenaemia with a gamma 275 Arg-->Cys substitution.

An abnormal fibrinogen was discovered in a clinically asymptomatic woman from Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times and a discrepancy between the plasma fibrinogen levels determined by the clotting assay and electroimmunoassay. Release of fibrinopeptides A and B from fibrinogen Milano V by thrombin was normal. Fibrin polymerization was strongly delayed in the presence of EDTA and was partially corrected at physiological calcium concentration. Normal migration of mercaptolysed polypeptide chains was observed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Moreover, there was no apparent abnormality in the charge of the reduced chains of the variant fibrinogen, as judged by two-dimensional gel electrophoresis. A fragment of the gamma-chain gene coding for the amino acids 259-350 was amplified and cloned. The amino acid gamma 275 arginine was found to be substituted by cysteine. Immunoblotting analysis with a rabbit antiserum against human serum albumin indicated that albumin was not linked to the odd sulphydryl group of fibrinogen Milano V. Treatment of fibrinogen Milano V with cysteamine, that is surmised to convert the mutant cysteine to a positively charged lysine analogue, did not improve the clotting properties of fibrinogen Milano V.

Fibrinogen Bern I: substitution gamma 337 Asn-->Lys is responsible for defective fibrin monomer polymerization.

An inherited fibrinogen variant, fibrinogen Bern I, was isolated from plasma of an asymptomatic woman. Routine coagulation studies showed prolonged thrombin and reptilase clotting times. Fibrinogen concentration was diminished when determined by a functional assay, but was normal by the heat precipitation method. The release of fibrinopeptides A and B was not delayed. Two-dimensional gel electrophoresis of mercaptolyzed fragments D of fibrinogen, obtained by digestion with plasmin, showed an abnormal electrophoretic mobility in the gamma-chain remnants of fragments D1 and D2 from fibrinogen Bern I, whereas conversion of D2 to D3 by plasmin resulted in the loss of the abnormal charge, suggesting that the structural abnormality in this variant is located in the region gamma 303 through 356. The molecular defect in fibrinogen Bern I was identified by sequence analysis of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet AAC coding for asparagine at position gamma 337 was found to be substituted by AAA coding for lysine. We conclude that the substitution gamma 337 Asn-->Lys in fibrinogen Bern I is responsible for defective polymerization of fibrin monomers and for impaired protection by calcium against plasmic degradation.

[Primary testicular tumors in childhood. Observations in 21 cases and a discussion of the necessity of metastasis prevention]. / Primäre Hodentumoren im Kindesalter. Beobachtungen an 21 Fällen und Diskussion der Notwendigkeit einer Metastasierungsprophylaxe.

A retrospective analysis of 21 primary testicular tumors in childhood is presented. Histologic review revealed 4 differentiated teratomas, 14 yolk sac tumors, 1 rhabdomyosarcoma of testicular envelopes and 1 paratesticular sarcoma. One tumor could not be classified. Two patients with yolk sac tumor and the patient with the paratesticular sarcoma died. In 4 of the 14 patients with yolk sac tumor iliac and/or paraaortic lymphnodes were removed 8-15 days after hemicastration but no tumor cells were found. Of 3 children with yolk sac tumor who developed metastases, one had had prophylactic resection and another one prophylactic irradiation of the draining lymphnodes. 8 patients with yolk sac tumor received prophylactic chemotherapy, and none developed metastases. For patients with yolk sac tumor prophylactic chemotherapy is indicated, particularly if more than 2 months have elapsed between the first detection of a testicular mass and operation. In prepubertal boys with testicular teratoma no prophylactic therapy to prevent dissemination is necessary. Patients with yolk sac tumor have an age distribution comparable to that of patients with an embryonal tumor.