Infra-inguinal arterial reconstruction for peripheral arterial disease: the Curaçao experience / Reconstrucción arterial infrainguinal en la enfermedad arterial periférica: la experiencia de Curazao

BACKGROUND: The prevalence and treatment of peripheral arterial disease in the Caribbean is not well documented. The aim of this study was to review the results from a small hospital in the Caribbean. METHODS: One-hundred and eight infra-inguinal arterial reconstructions on 90 patients were retrospectively reviewed Patients were classified according to the categories suggested by the Ad Hoc Committee on Reporting Standards of the Joint Councils of the Society for Vascular Surgery. Follow-up ranged from 0 to 103.1 months. The Kaplan-Meier method was used to visualize survival, limb salvage rates and primary and secondary patency rates. Cox regressions were used to identify potential risk factors. RESULTS: The limb salvage rates were 74.5% after one year and 71.4% after five years. Overall primary patency rates were 67.0 % after one year, 63.4% after three years and 50.8 % after five years. Overall secondary patency rates were 86.4% after one year and 75.1% after five years. The primary patency rate for autologous saphenous vein was 82.4% (SE 7.5%) after five years. The primary patency rates for prosthetic grafts were 62.1% (SE 8.5%) after one year; 56.9% (SE 9.2%) at three years and 37.9% (SE 16.7%) after five years. CONCLUSION: Infra-inguinal arterial bypass surgery is feasible in small Caribbean hospitals showing results comparable to major studies.

ANTECEDENTES: La prevalencia y el tratamiento de enfermedad arterial periférica en el Caribe no están bien documentados. El objetivo de este estudio fue examinar los resultados de un pequeño hospital en el Caribe. MÉTODOS: Se examinaron retrospectivamente ciento reconstrucciones arteriales infrainguinales en 90 pacientes. Los pacientes eran clasificados según las categorías sugeridas por el Comité Ad Hoc para el Reporte de Normas de los Consejos Unidos de la Sociedad de Cirugía Vascular. El seguimiento tuvo un rango de 0 a 103.1 meses. Se usó el método Kaplan-Meier con el objeto de ver las tasas de super-vivencia, salvamento de la extremidad, y tasas primarias y secundarias de permeabilidad. Se usaron regresiones de Cox para identificar los factores de riesgo potencial. RESULTADOS: Las tasas de salvamento de miembro fueron 74.5% después de un año y 71.4% después de cinco años. Las tasas generales de permeabilidad primaria fueron 67.0% después de un año, 63.4% después de tres años y 50.8% después de cinco años. Las tasas generales de permeabilidad secundaria fueron 86.4% después de un año y 75.1% después de cinco años. La tasa de permeabilidad primaria para la vena safena autóloga fue 82.4% (SE 7.5%) después de cinco años. Las tasas de permeabilidad primaria para los injertos prostéticos fueron 62.1% (SE 8.5%) después de un año, 56.9% (SE 9.2%) a los tres años y 37.9% (SE 16.7%) después de cinco años. CONCLUSIÓN: La cirugía de bypass arterial infrainguinal es factible en hospitales caribeños pequeños que muestran resultados comparables a los de estudios importantes.

Infra-inguinal arterial reconstruction for peripheral arterial disease: the Curaçao experience.

BACKGROUND: The prevalence and treatment of peripheral arterial disease in the Caribbean is not well documented. The aim of this study was to review the results from a small hospital in the Caribbean. METHODS: One-hundred and eight infra-inguinal arterial reconstructions on 90 patients were retrospectively reviewed Patients were classified according to the categories suggested by the Ad Hoc Committee on Reporting Standards of the Joint Councils of the Society for Vascular Surgery. Follow-up ranged from 0 to 103.1 months. The Kaplan-Meier method was used to visualize survival, limb salvage rates and primary and secondary patency rates. Cox regressions were used to identify potential risk factors. RESULTS: The limb salvage rates were 74.5% after one year and 71.4% after five years. Overall primary patency rates were 67.0 % after one year, 63.4% after three years and 50.8 % after five years. Overall secondary patency rates were 86.4% after one year and 75.1% after five years. The primary patency rate for autologous saphenous vein was 82.4% (SE 7.5%) after five years. The primary patency rates for prosthetic grafts were 62.1% (SE 8.5%) after one year; 56.9% (SE 9.2%) at three years and 37.9% (SE 16.7%) after five years. CONCLUSION: Infra-inguinal arterial bypass surgery is feasible in small Caribbean hospitals showing results comparable to major studies.

The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.

T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

T cell antigen-receptor signal transduction.

During the past year, major progress has been made in understanding proximal TCR signal-transduction events. Cbl has been identified as a negative regulator of kinases from the ZAP-70/Syk family. Studies on LAT, SLP-76, Itk and Vav have revealed their role in the activation of Ras and phospholipase-Cgamma1-Ca2+ signalling pathways. TCR-induced cytoskeletal changes involve signalling through SLP-76-Vav-Nck to activate effectors of the Rho-family of GTPases. Finally, glycolipid-enriched microdomains play a crucial role in T cell activation.

Activation of nuclear factor of activated T cells-(NFAT) and activating protein 1 (AP-1) by oncogenic 70Z Cbl requires an intact phosphotyrosine binding domain but not Crk(L) or p85 phosphatidylinositol 3-kinase association.

The Cbl proto-oncogene product is a complex adapter protein that functions as a negative regulator of protein tyrosine kinases. It is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 phosphatidylinositol 3-kinase (PI3K) upon engagement of numerous receptors linked to tyrosine kinases. Elucidation of the mechanism(s) underlying Cbl deregulation is therefore of considerable interest. The 70Z Cbl oncoprotein shows increased baseline tyrosine phosphorylation in fibroblasts and enhances nuclear factor of activated T cells (NFAT) activity in Jurkat T cells. Its transforming ability has been proposed to relate to its increased phosphotyrosine content. We demonstrate that 70Z Cbl shows increased basal and activation-induced tyrosine phosphorylation and association with Crk(L) and p85 PI3K in Jurkat T cells. 70Z Cbl, however, retains the ability to enhance NFAT and activating protein 1 (AP1) activity in the absence of Crk(L)/p85 PI3K association. In contrast, the G306E mutation, which inactivates the phosphotyrosine binding domain of Cbl, blocks NFAT/AP1 activation by 70Z Cbl. We conclude that 70Z Cbl-induced NFAT/AP1 activation requires the phosphotyrosine binding domain but not Crk(L)/p85 PI3K association. We hypothesize that 70Z Cbl acts as a dominant negative by blocking the negative regulatory function of the Cbl phosphotyrosine binding domain on protein-tyrosine kinases.

Calnexin association is not sufficient to protect T cell receptor alpha proteins from rapid degradation in CD4+CD8+ thymocytes.

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRalpha molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRalpha instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRalpha proteins in all other T cell types examined. Here, we compared the processing of TCRalpha glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRalpha glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRalpha proteins stably associated with calnexin in both cell types. Interestingly, however, TCRalpha association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+ thymocytes compared with splenic T cells. Finally, TCRalpha degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin.TCRalpha complexes. These data demonstrate that calnexin association is not sufficient to protect TCRalpha proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRalpha glycoproteins.

Reglucosylation of N-linked glycans is critical for calnexin assembly with T cell receptor (TCR) alpha proteins but not TCRbeta proteins.

Association of calnexin with newly synthesized glycoproteins involves recognition of monoglucosylated glycans, generated in the endoplasmic reticulum via initial removal of two glucose (Glc) residues from immature glycan chains by glucosidase enzymes (Glc trimming), or addition of a single Glc residue to fully trimmed glycans by glucosyltransferase enzymes (reglucosylation). While it has been established that creation of monoglucosylated glycans is important for chaperone binding, it is unknown if most proteins require both deglucosylation and reglucosylation for calnexin assembly or if initial Glc trimming is sufficient. Here, we studied the deglucosylation and reglucosylation of two related glycoproteins, the alpha and beta subunits of the T cell receptor (TCR) complex, and their assembly with calnexin in BW thymoma cells. Our data demonstrate that TCRalpha/beta glycoproteins undergo multiple cycles of Glc removal and addition within the endoplasmic reticulum and that numerous reglucosylated proteins assemble with calnexin, including TCRalpha/beta glycoproteins. Importantly, the current study shows that TCRbeta proteins, but not TCRalpha proteins, effectively associate with calnexin under conditions of functional Glc trimming but impaired reglucosylation. These data demonstrate that reglucosylated proteins associate with lectin-like chaperones in vivo and provide evidence that reglucosylation is of differential importance for the association of individual, indeed similar, glycoproteins with calnexin.

Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin-class I-TAP complexes.

Recent evidence indicates that newly synthesized major histocompatibility complex (MHC) class I proteins interact with calnexin, a transmembrane endoplasmic reticulum protein specific for certain glycoproteins bearing monoglucosylated glycans. Here, we studied the association of newly synthesized class I proteins with calreticulin, a soluble calnexin-related ER protein, in murine T cells. We found that, unlike calnexin-class I interactions, calreticulin assembly with class I proteins was markedly decreased in the absence of beta 2 microglobulin expression and that calreticulin associated with a subset of class I glycoforms distinct from those assembled with calnexin but similar to those bound to TAP (transporter associated with antigen processing) proteins. Finally, these studies show that deglucosylation of N-linked glycans is important for dissociation of class I proteins from both calreticulin and TAP and that the vast majority of newly synthesized class I proteins associated with calreticulin are simultaneously assembled with TAP. The data demonstrate that calnexin and calreticulin chaperones assemble with distinct MHC class I assembly intermediates in the ER and show that glycan processing is functionally coupled to release of MHC class I proteins from peptide transport molecules.

The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum.

Assembly of the multisubunit T cell antigen receptor (TCR) complex is an intricate process requiring coordinated regulation of at least six different gene products (alpha, beta, gamma, delta, epsilon, and zeta) and the ordered pairing of partner chains within the endoplasmic reticulum (ER). To date, two proteins have been implicated as functioning as molecular chaperones in the assembly of nascent TCR proteins: calnexin, a resident ER transmembrane protein, which associates with all TCR components except zeta, and T cell receptor-associated protein, which selectively associates with CD3gammaepsilon pairs. In this study, we examined the association of calreticulin, a soluble protein with significant sequence homology to calnexin, with newly synthesized TCR proteins. Analogous to calnexin, processing of glycan chains by glucosidase enzymes was required for initial association of TCRalpha and -beta proteins with calreticulin; however, several major differences were noted regarding interaction of calnexin and calreticulin chaperones with TCR proteins. First, TCRalpha and -beta proteins showed prolonged association with calnexin molecules compared with calreticulin; interaction of TCRalpha proteins with calreticulin was particularly transient, with most calreticulin-TCRalpha protein complexes dissociating within 15 min of their initial assembly. Second, we found that, unlike calnexin, which associated with clonotypic TCRalpha and -beta proteins and invariant CD3delta and -epsilon polypeptides, calreticulin associated specifically with clonotypic TCRalpha and -beta proteins. These studies identify calreticulin as a molecular chaperone for nascent clonotypic TCRalpha and -beta proteins and demonstrate that calreticulin and calnexin differentially associate with newly synthesized TCR proteins within the ER.

Calnexin associates exclusively with individual CD3 delta and T cell antigen receptor (TCR) alpha proteins containing incompletely trimmed glycans that are not assembled into multisubunit TCR complexes.

Most T lymphocytes express on their surfaces an oligomeric protein complex consisting of clonotypic alpha beta polypeptides associated with invariant CD3-gamma delta epsilon and zeta chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oligomerization of TCR alpha and CD3 delta glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3 delta proteins and TCR alpha proteins occurred prior to their association with any other TCR components and that calnexin specifically interacted with unassembled TCR alpha and CD3 delta proteins containing incompletely trimmed oligosaccharides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient association of calnexin with TCR alpha glycoproteins but not with CD3 delta glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3 delta and TCR alpha proteins occurring coincident with or immediately after their translocation into the endoplasmic reticulum and preceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3 delta and TCR alpha glycoproteins involves both glycan-dependent and glycan-independent mechanisms.

Relationship between patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency.

We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from 1968 until 1992. The patients received a graft from an HLA-identical related (n = 10), an HLA-haplo-identical related (n = 19), or a closely HLA-matched unrelated (n = 2) donor that resulted in the long-term survival of 6 of 10, 9 of 19, and 0 of 2 children, respectively. Major complications included failure of engraftment and early death caused by respiratory failure. The chimerism pattern and immunologic reconstitution were evaluated in 15 children who survived more than 1 year with sustained engraftment. The pattern of engraftment was investigated within flow-sorted peripheral blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain reaction. The immunologic reconstitution was assessed by various in vitro and in vivo parameters. Although the number of PB T cells and the in vitro T-cell proliferative response was in the lower region of normal in the majority of cases and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% to 5%) were detected in eight cases at multiple occasions. All children showed normal serum IgM and IgG subclass levels and produced specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical graft recipients with host-type B lymphoid and myeloid cells have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin. Donor NK cell engraftment restored defective in vitro NK cell function of the recipient. We conclude that determination of lineage-specific engraftment patterns provides valuable information for the understanding of the immunologic reconstitution after allogeneic BMT for SCID.

Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient's age and/or the conditioning regimen, but it is not associated with an increased risk of relapse.

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host-type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T-cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T-cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.

Mixed T-lymphoid chimerism after allogeneic bone marrow transplantation for hematologic malignancies of children is not correlated with relapse.

We performed polymerase chain reaction-variable number of tandem repeats analysis of flow-sorted peripheral blood T-, B-, natural killer-, and myeloid cell populations (van Leeuwen et al, Br J Haematol 79:218, 1991) in 32 children following allogeneic bone marrow transplantation (BMT) for leukemia to evaluate the relationship between mixed lymphoid chimerism and leukemia relapse. Five patients showed a stable mixed chimerism pattern characterized by the presence of both recipient as well as donor type cells in all cell populations up to 1 year posttransplantation. Five others showed transient mixed chimerism in the T-lymphoid cell lineage. In one patient, host T cells persisted until leukemia relapse. The remaining 21 patients showed a complete chimerism throughout the period of investigation. Twenty-five of these patients were classified according to the presence (n = 10) or absence (n = 15) of recipient type T cells. Statistical analysis did not show significant differences in the distribution of a number of clinical variables between the two groups, nor in the actuarial survival (P = .11) and leukemia-free interval (P = .97). Therefore, these results suggest that persistence of recipient type T lymphoid cells after allogeneic BMT for hematologic malignancies is not correlated with leukemia relapse. In addition, we observed that persistence of host cells within the original leukemia cell lineage and at the correct maturational stage was predictive for leukemia relapse in one case.

Detection of mixed chimaerism in flow-sorted cell subpopulations by PCR-amplified VNTR markers after allogeneic bone marrow transplantation.

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible. Flow sorted post-transplant peripheral blood B- and T-lymphocyte, natural killer and monocyte cell populations were subjected to PCR-VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.

Combined blunt rupture of the diaphragm and thoracic aorta.

Two patients with blunt traumatic rupture of the diaphragm and thoracic aorta are presented. This combination of injuries may be seen in the multiply-injured patient. Correct interpretation of chest radiographs is essential. If the diagnosis is made early the patient has a good chance of surviving. The eventual functional impairment is mainly related to the associated fractures.

Traumatic rupture of the diaphragm. Review of 62 successive cases.

From 1970 to 1987, 62 patients, suffering from traumatic rupture of the diaphragm, have been treated. In more than 75% of the cases, other severe post-traumatic associated lesions were noticed. In 54 cases these concerned blunt trauma, most of which were due to road accidents. There were 53 lesions of the left hemidiaphragm. Early diagnostics were made 40 times (i.e. within 24 hours after the accident). This is why it is all-important to correctly interpret the simple X-ray of thorax and abdomen, possibly after inserting a nasogastric tube. In general, exploration by laparotomy was preferred. Thoracolaparotomy or sternolaparotomy was only used in cases of thoracic or thoraco-abdominal injuries associated with important lesions at the lungs or the mediastinal organs; or in the case of long-standing ruptures accompanied by herniation of different organs. The total mortality rate was 21%. Death was due to hypovolemia, serious brain injuries or multiple organ failure (MOF) due to sepsis.