PubMed Health⌕ Search

Biomedical subjects

John E Connolly

Publications and source records attributed to John E Connolly.

18 recordsLinked to original sources
PubMed2006

Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

Amino Acid Sequence↗
PubMed2006

Aortoiliac endarterectomy: a lost art?

Endarterectomy was first performed on a superficial femoral artery in 1946 by Cid dos Santos and subsequently on the abdominal aorta by Wylie in 1951. During the 1950s and 1960s, aortoiliac endarterectomy (AIE) was the standard procedure for treatment of aortoiliac occlusive disease. When prosthetic graft material became available, aortobifemoral bypass (ABFB) replaced AIE in most cases because occlusive disease commonly affects the external iliac arteries also, which were difficult to endarterectomize. As a result, aorto-common iliac endarterectomy became almost a lost art. However, we believe there is still a place for AIE in selected patients based on a review of our results with the procedure. We reviewed 205 patients who survived 10 years after undergoing operation for aortoiliac occlusive disease by either aorto-common iliac endarterectomy (n = 39) or ABFB (n = 166). Ten-year primary patency was 89.2% for AIE and 78% for ABFB. Graft infection or aneurysmal formation occurred in 5% of ABFB and 0% of AIE cases. Ten male patients who underwent AIE for leg and hip claudication with positive penile/brachial indices of </=0.6 enjoyed improvement of erectile dysfunction. Twenty of the 39 AIEs were in female smokers with small vessels, localized disease, and elevated triglycerides. Three patients with end-to-side infected ABFB grafts, two with enteric fistula (one ours, two referred), had their grafts removed, followed by AIE with vein patching of their bypass sites. All three patients survived and at 10-year follow-up had patent reconstructed aortofemoral vessels. Since AIE avoids prosthetic material, it is preferable to ABFB in (1) patients whose aortoiliac occlusive disease does not involve the external iliac arteries; (2) male patients with aortoiliac occlusive disease who, in addition to claudication, have erectile dysfunction with penile/brachial indices of </=0.6 and stenotic internal iliac origins; (3) patients with aortoiliac disease including the external iliac arteries who are not candidates for ABFB because of infection risk or small vessels; (4) patients with localized aortoiliac disease; and (5) patients after removal of an infected ABFB graft (with or without an enteric fistula) that had initially been placed end-to-side for aortoiliac occlusive disease.

Aorta, Abdominal↗
PubMed2005

Structure/function analysis of tristetraprolin (TTP): p38 stress-activated protein kinase and lipopolysaccharide stimulation do not alter TTP function.

Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal; however, the mechanism by which TTP mediated RNA instability is unknown. Using an established model system, we performed structure/function analysis with TTP as well as examined the current hypothesis that TTP function is regulated by p38-MAPKAP kinase 2 (MK2) activation. Deletion of either the N- or C-terminal domains inhibited TTP function. Extensive mutagenesis, up to 16%, of serines and threonines, some of which were predicted to mediate proteasomal targeting, did not alter human TTP function. Mutation of the conserved MK2 phosphorylation sites enhanced human TTP function in both resting and p38-stress-activated protein kinase-MK2-activated cells. However, p38-stress-activated protein kinase-MK2 activation did not alter the activity of either wild-type or mutant TTP. TTP localized to the stress granules, with arsenite treatment reducing this localization. In contrast, arsenite treatment enhanced stress granule localization of the MK2 mutant, consistent with the involvement of additional pathways regulating this event. Finally, we determined that, in response to LPS stimulation, human TTP moves onto the polysomes, and this movement occurs in the absence of 14-3-3. Taken together, these data indicate that, although p38 activation alters TTP entry into the stress granule, it does not alter TTP function. Moreover, the interaction of TTP with 14-3-3, which may limit entry into the stress granule, is not involved in the downstream message stabilization events.

14-3-3 Proteins↗
PubMed2005

In situ saphenous vein bypass--forty years later.

In situ saphenous vein bypass, which was performed first by Rob in 1959, but introduced by Hall in 1962, has been widely applied as an alternative to the reversed bypass. Now, forty years later, it seems appropriate to review its current place and technique. Of the presumed original benefits of the in situ operation, it is now clear that the hemodynamic flow of converging (in situ) versus diverging vein (reversed) boundaries plus better vein/artery size match are the main advantages. It is now popular belief that the reversed saphenous vein graft to the popliteal artery has no significant hemodynamic disadvantage because the vein is of comparable diameter at the knee and in the groin. In contrast, vein bypass from the groin to the ankle strongly favors the in situ procedure because of the convergence of the walls of the vein below the knee and better vein/artery size match. The most controversial facet of the in situ operation has been the question of valvulotome is superior to lyse valves and whether to perform the operation open as originally described, or closed to avoid skin complications from a long groin to ankle incision. Preoperative vein mapping is advantageous for all saphenous vein conduit operations. For open leg in situ procedures, skin bridges and incisions made directly over the vein, directed by mapping, will minimize skin complications. Use of endoscopic in situ techniques is ideal but only with an experienced endoscopist. Valve lysis has improved but is still not foolproof.

History, 20th Century↗
PubMed2004

The role of mRNA turnover in the regulation of tristetraprolin expression: evidence for an extracellular signal-regulated kinase-specific, AU-rich element-dependent, autoregulatory pathway.

Tristetraprolin (TTP) is a regulator of TNF-alpha mRNA stability and is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. Using the THP-1 myelomonocytic cell line, we demonstrated for the first time that TTP is encoded by an mRNA with a short half-life under resting conditions. Using pharmacologic inhibitors of the mitogen-activated protein kinase pathways, we show that the induction of TTP by LPS activation is mediated through changes in transcription, mRNA stability, and translation. A coordinate increase in both TTP and TNF-alpha mRNA stability occurs within 15 min of LPS treatment, but is transduced through different mitogen-activated protein kinase pathways. This regulation of TTP and TNF-alpha mRNA stability is associated with the finding that TTP binds these mRNA under both resting and LPS-activated conditions in vivo. Finally, we demonstrate that TTP can regulate reporter gene expression in a TTP 3' untranslated region-dependent manner and identify three distinct AU-rich elements necessary to mediate this effect. Thus, TTP regulates its own expression in a manner identical to that seen with the TNF-alpha 3' untranslated region, indicating that this autoregulation is mediated at the level of mRNA stability. In this manner, TTP is able to limit the production of its own proteins as well as that of TNF-alpha and thus limit the response of the cell to LPS.

Cell Line↗
PubMed2004

A novel human cancer vaccine elicits cellular responses to the tumor-associated antigen, human chorionic gonadotropin beta.

PURPOSE: The oncofetal antigen, human chorionic gonadotropin beta subunit (hCGbeta), is expressed by a number of carcinomas and is a prognostic indicator in renal, colorectal, bladder, and pancreatic cancers. We describe the development of a novel antibody-based dendritic cell (DC)-targeted cancer vaccine capable of eliciting cellular immune responses directed against hCGbeta. EXPERIMENTAL DESIGN: The tumor-associated antigen hCGbeta was coupled genetically to a human anti-DC antibody (B11). The resulting fusion protein (B11-hCGbeta) was evaluated for its ability to promote tumor antigen-specific cellular immune responses in a human in vitro model. Monocyte-derived human DCs from normal donors were exposed to purified B11-hCGbeta, activated with CD40 ligand, mixed with autologous lymphocytes, and tested for their ability to promote hCGbeta-specific proliferative and cytotoxic T-lymphocyte responses. RESULTS: B11-hCGbeta was found to be a soluble, well-defined, and readily purified product that specifically recognized the human mannose receptor via the B11 antibody portion of the fusion protein. B11-hCGbeta functionally promoted the uptake and processing of tumor antigen by DCs, which led to the generation of tumor-specific HLA class I and class II-restricted T-cell responses, including CTLs capable of killing human cancer cell lines expressing hCGbeta. CONCLUSIONS: Although other hCG vaccines have been shown to be capable of eliciting antibody responses to hCGbeta, this is the first time that cellular immune responses to hCGbeta have been induced by a vaccine in a human system. This DC-targeted hCGbeta vaccine holds promise for the management of a number of cancers and merits additional clinical development.

Animals↗
PubMed2004

Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules.

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.

Amino Acid Sequence↗
PubMed2004

Differential expression of Toll-like receptors 2 and 4 in tissues of the human female reproductive tract.

Toll-like receptor (TLR) signal transduction is a central component of the innate immune response to pathogenic challenge. Although recent studies have begun to elucidate differences in acquired immunity in tissues of the human female reproductive tract, there is a relative paucity of work regarding innate defense mechanisms. We investigated TLR mRNA and protein expression in tissues of the human female reproductive tract. Constitutive mRNA expression of TLRs 1 to 6 was observed in fallopian tubes, uterine endometrium, cervix, and ectocervix. Furthermore, transcripts of the signaling adapter MyD88 and the accessory molecule CD14 were also detected in all tissues assayed. Quantitative analysis of TLR2 mRNA levels revealed highest expression of this molecule in fallopian tube and cervical tissues, followed by endometrium and ectocervix. In contrast to TLR2, TLR4 expression declined progressively along the tract, with highest expression in the upper tissues (fallopian tubes and endometrium), followed by cervix and ectocervix. In addition to mRNA, protein expression of TLR2 and TLR4 was also documented in these tissues. These data suggest that TLRs are differentially expressed in distinct compartments of the female reproductive tract and may provide insight regarding the regulation of inflammation and immunity within the tract.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Regulation and localization of endogenous human tristetraprolin.

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.

Antibody Specificity↗
PubMed2002

Lactate dehydrogenase is an AU-rich element-binding protein that directly interacts with AUF1.

Post-transcriptional pathways provide a major means of regulating eukaryotic gene expression. Reiterations of the AU-rich element (ARE) within the 3'-untranslated region of many cytokine and proto-oncogene mRNAs serve as signals for rapid degradation and translational repression. The identification of this cis-acting stability determinant has fueled the search for ARE-binding proteins (AUBP) that function as trans-acting factors that transduce this function. Previous work identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP capable of binding the ARE of granulocyte-macrophage colony stimulating factor (GM-CSF) RNA in the context of a full-length mRNA. We report here that functional studies failed to indicate a role for hnRNP A1 in ARE-dependent mRNA turnover. In an effort to identify other functionally relevant AUBP, the major GM-CSF ARE-specific binding protein in cells lacking hnRNP A1 was purified from CB3 mouse erythroleukemia cells. Microsequencing identified this protein as the glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was shown to occur in the NAD(+)-binding region (Rossmann fold). Polysome gradient analysis demonstrates that LDH is found in the translationally active fraction. Polysomal localization of LDH was dependent on RNA binding. Moreover, polysomal LDH exists in a complex with AUF1 and hsp-70, which has been implicated previously in the regulation of mRNA turnover. The interaction between LDH and AUF1 is direct as it can be demonstrated in vitro with purified proteins. Collectively these data implicate a role for LDH in the post-transcriptional regulation of gene expression.

Animals↗
PubMed2002

Analysis of the function, expression, and subcellular distribution of human tristetraprolin.

OBJECTIVE: The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNFalpha 3'-untranslated region (3'-UTR)-mediated expression. We also generated a specific polyclonal antibody against hTTP that enabled the examination of the subcellular distribution of hTTP and its RNA binding in vivo. METHODS: Transfection of reporter gene constructs were used to functionally characterize the role of hTTP in regulating TNFalpha expression in a 3'-UTR-dependent manner. An immunoprecipitation reverse transcription-polymerase chain reaction technique, immunoblotting, immunocytochemistry, and sucrose density fractionation were used to identify and localize hTTP. RESULTS: We found that hTTP interacted with human TNFalpha mRNA in the cytoplasm. The presence of the TNFalpha 3'-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNFalpha 3'-UTR adenine-uridine-rich element (ARE)-dependent manner. Immunoblotting and immunocytochemistry indicated that endogenous and transfected hTTP localized to the cytoplasm. Results of sucrose density fractionation studies were consistent with a polysomal location of hTTP. In rheumatoid synovium, hTTP expression was restricted to cells in the synovial lining layers. CONCLUSION: Through the development of an antiserum specific for hTTP, we have been able to demonstrate that hTTP binds specifically to the TNFalpha 3'-UTR and reduces reporter gene expression in an ARE-specific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNFalpha. Understanding the posttranscriptional regulation of TNFalpha biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis.

3' Untranslated Regions↗
PubMed2002

Middle aortic syndrome: distal thoracic and abdominal coarctation, a disorder with multiple etiologies.

BACKGROUND: Congenital coarctation of the thoracic aorta at the ligamentum arteriosum or the aortic arch is well recognized. But a much less common variety (0.5% to 2.0%) of aortic coarctation is located in the distal thoracic aorta, or abdominal aorta, or both and is often called "middle aortic syndrome" or "mid-aortic dysplastic syndrome." These types of aortic coarctation are most often secondary to a form of granulomatis vasculitis commonly known as Takayasu's disease in this country or aortitis syndrome in Japan. No single genesis explains every case and beside vasculitis as a cause, some are thought to be congenital in origin and others are associated with von Recklinghausen's disease. STUDY DESIGN: Eight patients with severe hypertension or claudication secondary to middle aortic coarctation were studied with aortograms and subsequently treated by vascular reconstruction procedures. RESULTS: Vascular reconstructions consisted of aortoaortic bypass, aortic resection with interposed grafting, reanastomotic resection of renal arteries into prosthetic grafts, and renal artery bypass with autogenous material. All eight patients' grafts have remained patent, with followups of 4 to 9 years, with relief of hypertension and claudication. Although Takayasu's disease can be progressive, aggressive surgical treatment in eight patients followed for 4 to 8 years postoperatively demonstrates that severe hypertension, claudication, or both are important indications for revascularization. CONCLUSIONS: Whatever the cause, assuming that active aortic inflammation has been medically treated and is in a burned-out state, patients with abdominal coarctation who have symptomatic renovascular hypertension, claudication, or both are good candidates for revascularization. Although surgical repair is more difficult than with congenital thoracic coarctation, because aortic walls are fibrotic and often also involve the renals, all eight of our patients had successful longterm correction of their hypertension and coarctation.

Adolescent↗
PubMedDate not supplied

Associations between static and functional measures of joint function in the foot and ankle.

Clinicians have traditionally assessed range of motion of the first metatarsophalangeal and ankle joints in a static position. It is unclear, however, if these measurements accurately reflect functional sagittal plane limitations of these joints during gait. For 50 patients (100 feet), we assessed available dorsiflexion at the first metatarsophalangeal and ankle joints, as well as the presence of pinch callus. We then compared these findings with 11 functional gait parameters, as measured using a pressure sensor system. After adjusting for age, weight, smoking status, glycosylated hemoglobin, and insensitivity to monofilament, we found that patients with pinch callus demonstrated statistically significant compensatory gait patterns in 7 of 11 measures. Hallux limitus and equinus patients demonstrated six and three statistically significant associations, respectively. Pinch callus seems to be as predictive of functional gait alterations as static first metatarsophalangeal joint and ankle dorsiflexion.

Aged↗
PubMedDate not supplied

Do clinical examination variables predict high plantar pressures in the diabetic foot?

Elevated plantar pressures are an important predictor of diabetic foot ulceration. The objective of this study was to determine which clinical examination variables predict high plantar pressures in diabetic feet. In a cross-sectional study of 152 male veterans with diabetes mellitus, data were collected on demographics, comorbid conditions, disease severity, neuropathy status, vascular disease, and orthopedic and gait examinations. Univariate predictors included height, weight, body surface area, body weight per square inch of foot surface area, bunion deformity, hammer toe, Romberg's sign, insensitivity to monofilament, absent joint position sense, decreased ankle dorsiflexion, and fat pad atrophy. Variables that remained significantly associated with high plantar pressures (>/=4 kg/cm(2)) in multivariate analysis included height, body weight per square inch of foot surface area, Romberg's sign, and insensitivity to monofilament. These results may be useful in identifying patients who would benefit from interventions designed to decrease plantar foot pressures.

Aged↗