Expression and mutation analysis of BRUNOL3, a candidate gene for heart and thymus developmental defects associated with partial monosomy 10p.

Partial monosomy 10p is a rare chromosomal aberration. Patients often show symptoms of the DiGeorge/velocardiofacial syndrome spectrum. The phenotype is the result of haploinsufficiency of at least two regions on 10p, the HDR1 region associated with hypoparathyroidism, sensorineural deafness, and renal defects (HDR syndrome) and the more proximal region DGCR2 responsible for heart defects and thymus hypoplasia/aplasia. While GATA3 was identified as the disease causing gene for HDR syndrome, no genes have been identified thus far for the symptoms associated with DGCR2 haploinsufficiency. We constructed a deletion map of partial monosomy 10p patients and narrowed the critical region DGCR2 to about 300 kb. The genomic draft sequence of this region contains only one known gene, BRUNOL3 ( NAPOR, CUGBP2, ETR3). In situ hybridization of human embryos and fetuses revealed as well as in other tissues a strong expression of BRUNOL3 in thymus during different developmental stages. BRUNOL3 appears to be an important factor for thymus development and is therefore a candidate gene for the thymus hypoplasia/aplasia seen in partial monosomy 10p patients. We did not find BRUNOL3 mutations in 92 DiGeorge syndrome-like patients without chromosomal deletions and in 8 parents with congenital heart defect children.

Propionibacterium acnes and inflammation in acne; P. acnes has T-cell mitogenic activity.

BACKGROUND: Circumstantial evidence suggests that Propionibacterium acnes has a role in the inflammation of acne. This could be effected by antigenic or superantigenic or mitogenic reactions. OBJECTIVES: The purpose of this investigation was to determine whether P. acnes had only antigenic activity or additional superantigenic and mitogenic activity. METHODS: A lymphocyte transformation assay was used to detect responses to a mixture of eight P. acnes whole cell isolates, and their supernatant culture fluids. In order to determine the nature of T-cell reactions to P. acnes cells a mouse-antihuman major histocompatibility complex class II monoclonal antibody was used in the lymphocyte transformation assay to inhibit the antigenic stimulation of lymphocytes. An analysis of the T-cell receptor (TCR) variable region beta (BV) repertoire was undertaken using flow cytometry of the unstimulated and stimulated cells. RESULTS: Peripheral blood mononuclear cells (PBMNC) from adults with no history of acne responded strongly to stationary growth phase cells of P. acnes, less strongly to cells in the exponential growth phase. No response was detected to supernatant culture fluids. PBMNC from five cord blood samples (CBMNC) responded maximally after 3 and 7 days of incubation with stationary growth phase cells of P. acnes. The reaction of CBMNC to P. acnes cells was not suppressed completely by the blocking antibody. The analysis of the TCRBV repertoire indicated that P. acnes induced no deletion or over-representation of certain BV element-bearing T cells. The TCRBV analysis was repeated after preincubation with the blocking antibody. Deletion of T cells bearing certain BV components occurred and there was no over-representation of T cells carrying certain BV components. CONCLUSIONS: Two mechanisms of lymphocyte activation by P. acnes cells are proposed, antigen and mitogen driven. These results are consistent with the histological evidence of inflammation in acne lesions.

Persistence of clonal T-cell expansions following high-dose chemotherapy and autologous peripheral blood progenitor cell rescue.

Analysing the regeneration of T lymphocytes after high-dose chemotherapy with autologous peripheral blood progenitor cell rescue (PBPCR) may help elucidate the mechanisms of immune recovery. The T-cell receptor variable beta chain (TCRBV) repertoire of adult patients undergoing high-dose chemotherapy was analysed by flow cytometry, before and after treatment. Four patients were found to have a stable expansion present (TCRBV3, 17, 21 and 22) ranging from 8% to 42% of the CD4(+) or CD8(+) repertoire. We demonstrated that, in these patients, following high-dose chemotherapy and autologous stem cell transplantation, the clonal expansions reappeared in peripheral blood and returned to pretransplant levels. Three expansions (CD3(+)CD8(+)TCRBV3(+), CD3(+)CD4(+)TCRBV21(+) and CD3(+)CD8(+)TCRBV22(+)) were further defined by sequence analysis of the complementarity-determining region (CDR)3 portion within the TCR rearrangements. These were shown to be predominantly clonal, with the same sequences being identified in peripheral blood before and after PBPCR, providing evidence that the overwhelming majority of T cells in these expansions arise from mature lymphocytes. This study demonstrated that patients undergoing autologous PBPCR for high-dose chemotherapy regenerate clonal expansions, consistent with pretreatment levels. They also regenerate T-cell repertoires with each TCRBV family represented to a similar level as that prior to high-dose chemotherapy.

Conserved TcR beta chain usage for a single MHC class II-restricted heat shock protein peptide.

Previously we have shown that the T-cell response against the HLA-DR3 (17)-restricted heat shock protein (Mr 65 000)-derived peptide amino acids (aa) 3-13 (hsp65 aa 3-13) is recognized by the exclusive usage of the TRBV5 gene as well as a conserved CDR3 region in a tuberculoid leprosy patient. In the present study we analyzed the TcR of T-cell clones specific for hsp65 aa 3-13 derived from three healthy individuals with a response level similar to that of the leprosy patient. We show that unlike the tuberculoid leprosy patient, healthy high responders have a diverse T-cell response to hsp65 aa 3-13. However, a striking observation was made: even though high responders have a diverse specific TcR repertoire, TRBV5-expressing clones from two healthy individuals could be isolated that were nearly identical to a dominant clone in the tuberculoid leprosy patient. In conclusion, the data show that restriction of TcR specific for an antigen correlates with the presence of that antigen in disease. However, the preferred TcR can also be detected in healthy high responders. A natural infection in vivo, as with the tuberculoid leprosy patient, may be responsible for the observed trimming and preferential outgrowth of a certain TcR.

House dust mite and cat allergen levels in domestic dwellings in Christchurch.

AIMS: To quantify the levels of Dermatophagoides pteronyssinus allergen (Der p I) and Felis domesticus allergen (Fel d I) in domestic dwellings in Christchurch and to assess possible relationships with housing characteristics. METHODS: Domestic dwellings (n = 93) were randomly selected and housing characteristics documented during the summer of 1994/95. Dust samples were obtained from the floor of the living room (LR) and bedroom (BR) and from the bed by standard vacuuming methods. The predominant mite species were determined and D pteronyssinus and F domesticus levels quantified. RESULTS: D pteronyssinus was the predominant (95%) species. D pteronyssinus allergen levels [geomean (95% confidence intervals) were 3.5(2.5-4.8) micrograms/g in LR, 10.1(7.5-13.7) micrograms/g in BR and 5.7(4.3-7.6) micrograms/g in the bed. F domesticus allergen levels were significantly higher (p < 0.001) in houses with cats than without cats [median (range) 93.2 (3.3-1227.2) micrograms/g and 2.9 (0.4-214.8) micrograms/g respectively]. Higher LR D pteronyssinus allergen levels were found in houses classified as having high indoor humidity and in houses situated in geographically damp locations. CONCLUSIONS: Domestic D pteronyssinus and F domesticus allergen levels in Christchurch are comparable with those found in other climatically similar locations. D pteronyssinus allergen levels are associated with both indoor and outdoor humidity factors.

Economic evaluation of allogeneic bone marrow transplantation: a rudimentary model to generate estimates for the timely formulation of clinical policy.

PURPOSE: To provide an evidence-based approach to the formulation of clinical policy with respect to allogeneic bone marrow transplantation (BMT) that involves perceived trade offs between two major factors: costs and consequences. The report also highlights key informational deficiencies. PATIENTS AND METHODS: Adults with acute myeloid leukemia (AML) in second complete remission (2CR) and those with acute lymphoblastic leukemia (ALL) in first complete remission (1CR) were assigned to BMT or control groups solely on the availability of a suitable donor. All hospital-borne costs were estimated, based on services used according to manual chart review, in four categories: diagnostic and therapeutic costs, professional fees, drug costs, and ward costs. Incremental costs and incremental life-years were calculated, and the quotient determined a cost per life-year gained by BMT for AML (2CR) and ALL (1CR). RESULTS: The incremental cost (in 1992 Canadian dollars) per life-year gained by BMT (cost-effectiveness) for AML (2CR) was $29,200; and for ALL (1CR) it was minus $29,200. CONCLUSION: For AML (2CR), allogeneic BMT creates better outcomes than standard treatment, but is more costly. For ALL (1CR), both the costs and outcomes are similar for BMT and standard therapy. Quality adjustments made to life-years gained did not change these conclusions.

Human T cell repertoire generation in the absence of MHC class II expression results in a circulating CD4+CD8- population with altered physicochemical properties of complementarity-determining region 3.

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.

Marked conservation of complementarity-determining region 3 of the beta-chain of TCRs recognizing a mycobacterial heat shock protein 60-derived peptide with strong sequence similarity to human heat shock protein 60.

The variable gene usage and sequence of human TCRs specific for a particular MHC/peptide combination have been investigated. The peptide comprises amino acids 456-466 of the 65-kDa Mycobacterium tuberculosis heat shock protein (hsp60), and is recognized in the context of HLA-DP. TCRs from both synovial fluid and peripheral blood (PB)-derived T cell clones used only five different V beta genes, three of which are closely related (V beta 6.7a, V beta 6.7b, and V beta 21.3). Among TCRs using these three genes there was marked conservation of the beta-chain sequence, whereby complementarity-determining region 3 (CDR3) contained an amino acid motif (*R*G*, amino acids 96-100) in association with either J beta 1.4 or J beta 2.5. These conclusions were strengthened by analysis of peptide-stimulated T cell lines that revealed not only TCR beta-chain sequences identical with those seen in T cell clones, but also additional beta-chains with similar CDR3 region sequences and J gene usage. In contrast, T cell lines derived by using IL-2 or a control peptide revealed variable usage of V beta and J beta genes; V beta 6.7a/b sequences from these lines and from freshly isolated PB did not contain the CDR3 motif noted in TCRs from Ag-specific T cells. We suggest that the remarkably limited diversity of TCRs noted in this study is a consequence of the similarity between the mycobacterial hsp60 peptide and the equivalent peptide from human hsp60, and reflects the trimming of the TCR repertoire required to maintain self-tolerance.

Identification of the epitope recognized by the human V beta 5-specific monoclonal antibody 42/1C1. Potential implications for disease therapy.

A panel of T-cell clones was generated that was specific for amino acid residues 4-13 of the mycobacterial 65-kd stress protein. All the clones were found to express a member of the V beta 5 family, as defined by PCR. However, the clones could be differentiated on the basis of different staining characteristics with the mAb 42/1C1. This antibody is known to recognize both V beta 5.2 and V beta 5.3, as was the PCR primer pair used in the analysis. Sequencing of the TCRs revealed that those clones which were not stained by 42/1C1 expressed a previously unidentified member of the V beta 5 family. By comparing the sequences of the V beta 5 family members that are recognized by 42/1C1 with those that are not, we were able to identify a probable epitope for the antibody. It is also clear from our data that the TCRs of T cells recognizing identical MHC-peptide combinations, although very similar, may be differentiated by mAbs, thereby posing potential problems in any proposed disease therapy involving treatment with monoclonals.

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: selective in vivo expansion of T cells bearing defined receptors.

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either V beta 5.2 or a closely related beta chain, V beta 5.6. The alpha chains expressed by V beta 5.2+ and V beta 5.6+ clones were from different families, V alpha 2.4 and V alpha 23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either V beta 5.2 or V beta 5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of V beta 5. Conservation of the beta chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing alpha and beta chains of representative V beta 5.2+ and V beta 5.6+ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of alpha chain usage, since all clones expressed a member of the V alpha 1 family, but again CDR3 sequence conservation was not apparent. beta chain usage was not restricted since different clones expressed V beta 6.7, V beta 22.3 and V beta 12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical alpha and beta TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.

Intravenous streptokinase. A reappraisal of its therapeutic use in acute myocardial infarction.

Streptokinase, the first of the thrombolytic agents to be used in acute myocardial infarction, has now been administered to many thousands of patients with this condition. Since early intervention and accessibility of care is paramount in these patients, intravenous infusion of streptokinase has largely replaced intracoronary use. Results of major trials (GISSI, ISIS-2 and ISAM) comparing streptokinase with standard treatment in more than 30,000 patients prove convincingly that intravenous streptokinase increases patient survival after myocardial infarction. The largest trial, ISIS-2, demonstrated a 23% reduction in 5-week vascular mortality after streptokinase use. The greatest benefits occur where streptokinase infusion is initiated early after symptom onset, although late benefit has been observed in patients treated up to 24 hours after pain onset. Importantly, mortality is further decreased by combining streptokinase with aspirin, as shown by a 53% reduction in mortality using the combination in the ISIS-2 trial. Mortality has also been reduced in trials investigating the use of the thrombolytic agents rt-PA and anistreplase. Streptokinase and rt-PA produced similar reductions in mortality in the recent GISSI-2 and International t-PA/Streptokinase Mortality trials, findings which may be further clarified by ongoing comparative trials such as ISIS-3. Reperfusion of about 50 to 60% of occluded coronary arteries occurs with intravenous streptokinase, and left ventricular function is improved. Direct comparisons with rt-PA show a superior effect for the newer agent on early reperfusion, but a similar ability to salvage myocardial function. The complexities of the relationship between reperfusion, left ventricular function and mortality constitute an area of considerable clinical interest requiring further study to clearly differentiate between the drugs available to the physician. The most common adverse events observed during intravenous streptokinase infusion are bleeding complications. An incidence of 3.6% for minor bleeding and 0.4% for major haemorrhage (requiring transfusion) is derived from the combined results of the GISSI and ISIS-2 studies. Bleeding does not appear to be more frequent or severe with intravenous streptokinase than with the more fibrin-selective agent, rt-PA. While the risk to benefit ratio of sequential heparin following streptokinase therapy remains equivocal, the adjuvant use of aspirin confers a clinical advantage over streptokinase alone. In conclusion, streptokinase has now been proven to reduce mortality in patients with acute myocardial infarction, with an acceptable risk of bleeding complications. Given the substantial data that have now accumulated with extensive clinical experience, intravenous streptokinase should be considered a first-line agent in suitable patients.