John E. Connolly

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

ABSTRACT 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.

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.

A Novel Human Cancer Vaccine Elicits Cellular Responses to the Tumor-Associated Antigen, Human Chorionic Gonadotropin β

Purpose: The oncofetal antigen, human chorionic gonadotropin β subunit (hCGβ), 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 hCGβ. Experimental Design: The tumor-associated antigen hCGβ was coupled genetically to a human anti-DC antibody (B11). The resulting fusion protein (B11-hCGβ) 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-hCGβ, activated with CD40 ligand, mixed with autologous lymphocytes, and tested for their ability to promote hCGβ-specific proliferative and cytotoxic T-lymphocyte responses. Results: B11-hCGβ 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-hCGβ 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 hCGβ. Conclusions: Although other hCG vaccines have been shown to be capable of eliciting antibody responses to hCGβ, this is the first time that cellular immune responses to hCGβ have been induced by a vaccine in a human system. This DC-targeted hCGβ vaccine holds promise for the management of a number of cancers and merits additional clinical development.

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 thiscis-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.

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.

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.

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, Rombergs 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, Rombergs sign, and insensitivity to monofilament. These results may be useful in identifying patients who would benefit from interventions designed to decrease plantar foot pressures.

Clinical factors associated with a conservative gait pattern in older male veterans with diabetes

BackgroundPatients with diabetes and peripheral neuropathy are at higher risk for falls. People with diabetes sometimes adopt a more conservative gait pattern with decreased walking speed, widened base, and increased double support time. The purpose of this study was to use a multivariate approach to describe this conservative gait pattern.MethodsMale veterans (mean age = 67 years; SD = 9.8; range 37–86) with diabetes (n = 152) participated in this study from July 2000 to May 2001 at the Veterans Affairs Medical Center, White River Junction, VT. Various demographic, clinical, static mobility, and plantar pressure measures were collected. Conservative gait pattern was defined by visual gait analysis as failure to demonstrate a heel-to-toe gait during the propulsive phase of gait.ResultsPatients with the conservative gait pattern had lower walking speed and decreased stride length compared to normal gait. (0.68 m/s v. 0.91 m/s, p < 0.001; 1.04 m v. 1.24 m, p < 0.001) Age, monofilament insensitivity, and Rombergs sign were significantly higher; and ankle dorsiflexion was significantly lower in the conservative gait pattern group. In the multivariate analysis, walking speed, age, ankle dorsiflexion, and callus were retained in the final model describing 36% of the variance. With the inclusion of ankle dorsiflexion in the model, monofilament insensitivity was no longer an independent predictor.ConclusionOur multivariate investigation of conservative gait in diabetes patients suggests that walking speed, advanced age, limited ankle dorsiflexion, and callus describe this condition more so than clinical measures of neuropathy.

Primary closure of infected diabetic foot wounds. A report of closed instillation in 30 cases

Multiple surgical strategies are available for managing the infected diabetic foot at risk for amputation. The authors present their experience with the closed instillation system in the management of 30 such cases in 29 patients over a 5-year period. Data were collected from the hospital records of neuropathic patients presenting with deep-plantar-space infections or presumed acute osteomyelitis. All 29 patients were male; 57% had marginal or poor vascular supply, and 83% were nutritionally compromised or had proteinuria. At the conclusion of the study, 34% of the patients were dead, reflecting the severity of comorbid conditions found in this population. Despite the marginal healing capacity of these patients, the procedure had a 90% success rate, as defined by expeditious return to prior level of functioning and residential living situation without need for re-operation or higher-level amputation.

Intraosseous epidermoid inclusion cyst presenting as a paronychia of the hallux.

Intraosseous epidermoid inclusion cysts are rare benign bone lesions that occur most commonly in the skull and in the distal phalanges of the fingers. Herein we report a case of an intraosseous epidermoid inclusion cyst occurring in the distal phalanx of the left hallux. Only six occurrences of this lesion have been described in the foot. This patients presentation, with active drainage (initially appearing as purulent discharge from an acutely tender ingrown hallux nail) and a known inoculation event accompanied by severe peripheral vascular disease, make this case unique.