Familial antithrombin III deficiency in a Malay patient with massive thrombosis.

Patients with low antithrombin III (AT III) has increased risk for arteriovenous thromboembolic (TE) disease. We report a 28-year-old Malay lady who presented with spontaneous right calf pain and swelling of one week duration. She was on oral contraceptive pills and had a history of travelling for a long distance prior to the presentation. Her brother who was diagnosed with AT III deficiency had arterial thrombosis at a young age. She was diagnosed as having right popliteal vein thrombosis by ultrasound and treated with subcutaneous fondaparinux. While on treatment, she developed massive bilateral pulmonary embolism (PE). Thrombophilia study showed reduced AT III activity (38µl/dl) and normal results for protein C, protein S, activated protein C resistance and lupus anticoagulant assays. This patient has heterozygous AT III deficiency added with significant acquired factors responsible for the TE events. Those with AT III deficiency may have resistance to heparin therapy and require higher doses of heparin.

Von Hippel - Lindau disease.

Von Hippel-Lindau (VHL) disease is a rare autosomal dominantly inherited multisystem disorder characterised by the development of a variety of benign and malignant tumours. We report a case of VHL disease that was inherited by a daughter from her father, who both presented at a young age with progressive headache and were found to have a posterior fossa haemangioblastoma (HB) on magnetic resonance imaging (MRI). Multiple benign pancreatic and renal cysts were also noted in both patients.

Von Hippel–Lindau disease

Von Hippel–Lindau (VHL) disease is a rare autosomal dominantly inherited multisystem disorder characterised by the development of a variety of benign and malignant tumours. We report a case of VHL disease that was inherited by a daughter from her father, who both presented at a young age with progressive headache and were found to have a posterior fossa haemangioblastoma (HB) on magnetic resonance imaging (MRI). Multiple benign pancreatic and renal cysts were also noted in both patients.

Effects of copper overload on hepatic lipid peroxidation and antioxidant defense in rats.

The influence of copper (Cu) overload on hepatic lipid peroxidation and antioxidation defense capacity was studied by overloading rats with copper sulphate orally (500 mg Cu/kg bw) 5 d/w for 8 w. Malondialdehyde (MDA), Cu-Zn superoxide dismutase (SOD), and Se-glutathione peroxidase (GSH-Px) were measured in serum and liver homogenate at 2, 4 and 8 w of dosing. Liver Cu concentration and alanine aminotransferase (ALT) activity were also determined. As Cu loading progressed, there were multiparameter changes with significant ALT elevation, increased MDA concentrations in serum and liver homogenate, and dramatic declines of SOD and GSH-Px activities in erythrocytes and whole blood respectively, along with marked elevation of hepatic Cu in the Cu-dosed group. Excessive Cu accumulation in the liver depressed SOD and GSH-Px activities and resulted in high MDA in serum and liver homogenate due to the lipid peroxidation induced by the Cu overload.

The concentration of selected minerals and phytate in Malaysian Brachiaria decumbens.

Samples of Brachiaria decumbens collected from 5 farms representing the Peninsular Malaysia were subjected to selected trace mineral and phytate analyses to explain the pathogenesis of B decumbens intoxication. Concentrations of Cu, Zn, Fe and Mo were comparable to other grasses while that of phytate was low. The molar ratios of Cu:Zn, Cu:Mo, and Cu:Fe warrant that Cu deficiency is involved in the toxicity of B decumbens. This might aggravate the development of photosensitization of unpigmented or lightly pigmented areas of affected animals. The Zn:phytate ratio could predispose to Zn deficiency during intoxication.

Mapping major and minor T-cell epitopes in vitro and their immunogenic or tolerogenic effect in vivo in non-human primates.

The immunogenicity of synthetic peptides of in vitro mapped T- and B-cell epitopes from a Streptococcus mutans cell-surface antigen were investigated in non-human primates. Peptide (1-15) contains T-cell (7-15) and B-cell (8-13) epitopes, but is only immunogenic if dimerized (1-15)2 or linked to the carrier tetanus toxoid (1-15)TT. Monomers and dimers of T- and B-cell epitopes were prepared and used to immunize macaques. Immunogenicity was assayed in lymphocytes by the uptake of [3H]thymidine and serum antibodies by a solid-phase radioimmunoassay. Macaques immunized with the dimerized (1-15)2 or carrier-linked peptide (1-15)TT exhibited in vitro T-cell proliferative responses to peptides (1-15) and (7-15). T cells from animals immunized with peptides (1-15), (7-15) or (7-15)2 failed to elicit an immune response. In order to establish if these non-immunogenic peptides might induce tolerance, the same macaques were challenged with the immunogenic peptide (1-15)TT. The results suggest that T-cell responses to peptide (1-15) were reestablished, but instead of responding to peptide (7-15) they were stimulated by a hitherto silent epitope (1-7). Tolerance to the major T-cell epitope (7-15) and the expression of a minor (silent) T-cell epitope (1-7) was associated with B-cell tolerance, suggesting that T-cell help for antibodies resides in the major T-cell epitope (7-15). However, short-term T-cell lines revealed T-cell responses to peptides (1-7) and (7-15) in both tolerized and immunized macaques, but the relative frequency of the minor epitope (1-7)-reactive lines was significantly higher in tolerized animals, whilst that for the major epitope (7-15) was higher in immunized animals. These findings suggest that the silent epitope (1-7) is really cryptic, in that it can be detected if the cell lines are first expanded in vitro with the whole peptide (1-15) and then stimulated with the truncated peptides (1-7) or (7-15). The results are consistent with the concept of a hierarchy of major and minor T-cell epitopes, now demonstrated in non-human primates, in which tolerance to the major T-cell epitope is associated with tolerance to antibody formation and the emergence of a minor T-cell epitope.

Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.

An analysis of synthetic peptide restriction by HLA-DR alleles in T cells from human subjects, naturally sensitized by Streptococcus mutans.

T cells from most human subjects show significant in vitro proliferative responses to a 185-kDa surface Ag from Streptococcus mutans as well as to synthetic peptides derived from the sequence of a Mr 3800 streptococcal Ag. T cells from subjects expressing each of the alleles from DR1 to DR7 responded to synthetic peptides of 17 or 21 amino acid residues. Furthermore, inhibition studies with mAb to HLA class I and class II Ag showed that the DR Ag was a restriction molecule for the proliferative responses. Mouse L cells transfected with DR1, DR2, DR4, DR5, and DR7 were used to confirm the permissive nature of the responses. An analysis of the fine specificity of the responses showed that the minimum peptides capable of stimulating T cells from subjects with different DR types varied by one or two residues. For DR2 and DR3 the shortest peptide was residues 6-15, an additional serine (residue 5) was required for DR1 and DR7 and an aspartic acid (residue 4) for DR4, DR5, and DR6. Successful oral-mucosal bacterial colonisation in humans, by a largely commensal Streptococcus, might be associated with the permissive nature of the HLA-DR restriction of the response to a major streptococcal cell surface peptide. The peptide recognised in association with the HLA-DR molecule may induce an immune response that prevents central entry of the organism from the peripheral mucosal site.

The comparative frequency of human T cells responding to a native antigen and a synthetic peptide derived from Streptococcus mutans.

The frequency of human peripheral blood T cells responding to a 21-residue synthetic peptide (SP 21) derived from the sequence of a 3.8-kDa streptococcal antigen was estimated by limiting dilution analysis and compared with the frequency of cells responding to the native, cross-reactive 185-kDa streptococcal antigen. Frequency estimates were made by measuring both [3H]thymidine incorporation and IL 2 production in the same cell cultures. The results provided frequency estimates for SP 21-reactive cells of between 1:42 147 and 1:306 110, with a mean of 1:160 617 by [3H]thymidine incorporation, and 1:139 893 to 1:241 315 (mean 1:165 315) using the IL 2 assay. With the native 185-kDa streptococcal antigen, frequency estimates were between 1:38 393 and 1:86 142 (mean 1:169 934) according to the proliferative response and 1:22 462 and 1:100 400 (mean 1:61 189) by the IL 2 assay.

The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.

Identification of T- and B-cell epitopes in synthetic peptides derived from a Streptococcus mutans protein and characterization of their antigenicity and immunogenicity.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 Mr Streptococcus mutans antigen was found in human subjects. Significant serum IgG antibodies were detected both to the native streptococcal antigen and to the SP17, containing essentially residues 1-15. A series of short peptides with deletions at the amino- and carboxy-termini were then tested to identify the B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies but only the former consistently inhibited human antibodies, suggesting that residues 8-13 constitute a major B-cell epitope. The human CD4 subset of T-cells was then examined and this showed a significant uptake of [3H]-thymidine when stimulated with both the native streptococcal antigen and the SP17. The series of short peptides was then used to stimulate CD4 cells, in order to determine the T-cell epitope. The synthetic peptide with residues 6-15 was the shortest peptide that stimulated significant [3H]-thymidine uptake and this peptide was designated as a T-cell epitope. The immunogenicity and antigenicity of SP17 was also investigated in macaques. Immunization of monkeys with the free SP17 failed to elicit serum antibodies or T-cell responses. However, immunization with SP17 linked to tetanus toxoid as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the synthetic peptide but also to the native streptococcal antigen. As in the human studies a B-cell epitope was found in residues 8-13, whereas an overlapping T-cell epitope was located in residues 7-15.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of synthetic peptides derived from the sequences of a Streptococcus mutans cell surface antigen in nonhuman primates.

The immunogenicity and antigenicity of synthetic peptides (SP) derived from the sequences of a cell surface Ag of Streptococcus mutans were investigated in macaque monkeys. Immunization with the free peptides of 11, 17, and 21 residues failed to elicit serum antibodies or T cell responses. However, immunization with the SP17 and SP21 linked to tetanus toxoid (TT) as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the SP but also to the native streptococcal Ag. In vivo recall of SP-TT immunized monkeys with suboptimal doses of the native streptococcal Ag resulted in a significant increase in antibodies, both to the SP and the streptococcal Ag, confirming that the SP shares antigenic epitopes with the native Ag. B and T cell epitopes were then determined and a B cell epitope was found in residues 8-13, whereas an overlapping T cell epitope was located in residues 7-15. The T cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residues 8-13 of the B cell epitope. In spite of the B and T cell epitopes being expressed in SP17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. However, immunization with a dimer of SP17 elicited both serum antibodies and proliferative responses of lymphocytes without a carrier. The results suggest that the monomeric SP17 is not immunogenic and needs to be dimerised in order to elicit antibodies and T cell responses, both to the SP and to the streptococcal Ag.

Local oral immunization with synthetic peptides induces a dual mucosal IgG and salivary IgA antibody response and prevents colonization of Streptococcus mutans.

A small cell surface antigen of Streptococcus mutans was partially sequenced and the amino terminal peptides of 11, 15 and 20 amino acid residues and a dimer of the 15 and 20 residues peptides were synthesized. The synthetic peptides (SP) were used in topical oral immunization of the gingivomucosal epithelium of macaque monkeys. Sequential examination for antibodies over a period of up to 30 weeks revealed that six applications of the linear or cyclized SP11 and a random SP11 induced negligible or very low antibody levels. In contrast, the SP17 (SP15 with added cysteine at each terminus), SP21 (SP20 with one cysteine) and the dimer (SP35) induced significant anti-SP as well as anti-native streptococcal antibodies in the gingival fluid and in saliva. The functional significance of this immune response was examined by studying its effect on oral colonization of S. mutans following feeding of a carbohydrate-rich diet. Whereas control animals, sham-immunized with a random SP of 11 residues, showed increased colonization of the teeth by S. mutans, there was no colonization or a significant reduction in colonization of animals immunized with the cyclized SP17, linear SP21 or dimerized SP35. These experiments suggest that local immunization with SP derived from the sequences of a streptococcal cell surface antigen induce a dual local immune response of gingival IgG and salivary IgA antibodies against the SP and native SA. These antibodies may be involved in preventing colonization of S. mutans, which is the principal agent in the development of dental caries.

T cell interactions generated by synthetic peptides covalently linked to a carrier.

We have attempted to extend the synthetic peptide-carrier bridge concept of T cell-B cell interaction to T cell-T cell interaction. DNA synthesis of human CD4 cells that were sensitized in vivo to a native streptococcal antigen (SA) was stimulated in vitro with synthetic peptides (SP) derived from the sequence of native SA. The SP were linked to tetanus toxoid (TT) as a carrier which was recognized by primed T cells. The uptake of [3H]thymidine was significantly greater when stimulated with covalently linked SP-TT than that with non-covalently mixed SP and TT. The TT- and SP-sensitized CD4 cells were then enriched and depleted by panning on TT- or SP-treated monocyte layers. When TT-enriched CD4 cells were reconstituted with SP-enriched cells, [3H]thymidine uptake was significantly greater with the linked SP-TT than with the mixed SP and TT. However, reconstitution of the TT-enriched with SP-depleted CD4 cells or the converse failed to increase significantly DNA synthesis by cells stimulated with the linked SP-TT. The production of interleukin 2 (IL 2) and expression of IL 2 receptors were then assayed to examine any difference in stimulation between TT and SP. Both IL2 and IL2 receptors were diminished and delayed when T cells were stimulated with SP as compared with TT. The results suggest that epitope-linked clusters of monocytes, TT-sensitized CD4 and SP-sensitized CD4 cells enable IL2 released by the TT-sensitized CD4 cells to stimulate the SP-sensitized CD4 cells that produce inadequate amounts of IL2. Indeed, addition of recombinant IL2 to T cells stimulated with mixed SP and TT induces an increase in DNA synthesis which becomes similar to that resulting from stimulation with the linked SP-TT.

Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.