Limited Audiological Assessment Results in Children With Otitis Media With Effusion.

OBJECTIVES: Clinical practice guidelines predicate the need for evaluation of hearing in children with otitis media with effusion (OME). The objective of this work was to characterize the completeness of hearing assessment results in children with OME. DESIGN: Forty participants with OME completed two full audiological assessments, one in a clinical setting and a second in a research setting. An additional 14 participants without OME completed a single audiological assessment in the research setting as a control group. The success of various behavioral and objective audiometric tests in each setting was quantified and evaluated. RESULTS: Findings indicate that ear-specific behavioral audiometric information is substantially limited in children with OME, particularly in clinical settings. In contrast, objective testing including tympanometry and otoacoustic emission testing was largely successful. CONCLUSIONS: Ear-specific behavioral audiometric information is limited in children with OME and, consequently, consideration of these data for use as part of clinical decision making is also limited. Objective tests were more successful but are not direct measures of hearing.

Audiologic Profiles of Children With Otitis Media With Effusion.

OBJECTIVES: To describe the impact of effusion volume, viscosity, and purulence on the audiologic profiles of children with otitis media with effusion. DESIGN: Fifty-one ears from children between the ages of 8 months and 11 years who had a diagnosis of otitis media with effusion and were scheduled for tympanostomy tube placement were recruited from medical clinics. The control group consisted of 17 ears from children between the ages of 10 months and 11 years without a recent history of otitis media and were recruited from a database of research volunteers. Participants received a comprehensive audiologic testing battery consisting of tympanometry, otoacoustic emissions, behavioral audiometric thresholds, and auditory brainstem response testing. For children with otitis media, this testing battery occurred 1 to 2 days before surgery. Middle ear effusions were characterized and collected on the day of surgery during tympanostomy tube placement from ears with otitis media with effusion. The comprehensive audiologic testing battery was completed postoperatively as well for most participants. RESULTS: Effusion volume, categorized in each ear as clear, partial, or full, effected the audiologic results. Ears with full effusions had moderate hearing losses, few to no measurable otoacoustic emissions, and delayed Wave V latencies. Ears with partial effusions and clear ears both had slight to mild hearing losses and normal Wave V latencies, though ears with partial effusions had fewer measurable otoacoustic emissions than clear ears. Normal-hearing control ears with no recent history of otitis media with effusion demonstrated normal audiometric thresholds, present otoacoustic emissions, and normal Wave V latencies. Repeat postoperative testing demonstrated improvements in audiologic testing results for all of the otitis media with effusion volume groups, with no significant differences remaining between the three otitis media with effusion groups. However, significant differences between otitis media with effusion ears and normal-hearing control ears persisted postoperatively, with otitis media with effusion ears demonstrating significantly poorer audiometric thresholds and reduced otoacoustic emissions as compared to normal control ears. The effect of effusion viscosity and purulence could not be systematically evaluated because minimal variability in effusion viscosity and purulence was observed in our sample, with nearly all effusions being mucoid and nonpurulent. CONCLUSIONS: Effusion volume observed at the time of tympanostomy tube surgery was found to play a significant role in outcomes and responses on a range of audiologic tests that compose the standard clinical pediatric audiologic assessment battery. Full middle ear effusions were associated with a moderate hearing loss, and few to no measurable otoacoustic emissions were detected. Ears with a recent diagnosis of otitis media with effusion but clear at the time of tympanostomy tube placement had less hearing loss and a greater number of present otoacoustic emissions than ears with full or partial effusions but were still found to have poorer hearing sensitivity than the healthy control ears. Differences between ears with otitis media with effusion and healthy control ears persisted on postoperative assessments of otoacoustic emissions and audiometric thresholds, though there were no remaining effects of the presurgical effusion volume group.

Improving the Differential Diagnosis of Otitis Media With Effusion Using Wideband Acoustic Immittance.

OBJECTIVES: The objective of this work is to determine whether there is a systematic effect of middle ear effusion volume on wideband acoustic immittance in children with surgically confirmed otitis media with effusion. DESIGN: Wideband acoustic immittance was measured in 49 ears from children (9 months to 11 years) who had a diagnosis of otitis media with effusion and compared to 14 ears from children (10 months to 10 years) without a recent history of otitis media. For children with otitis media with effusion, wideband acoustic immittance testing took place in the child's preoperative waiting room before surgical placement of tympanostomy tubes. Testing was completed in a pressurized condition (wideband tympanometry) for all ears as well as in an ambient condition in a subset of ears. Intraoperative findings regarding effusion volume were reported by the surgeons immediately before tube placement and confirmed following myringotomy. This classified the volume of effusion as compared to middle ear volume categorically as either full, partial, or clear of effusion. The type of wideband acoustic immittance explored in this work was absorbance. Absorbance responses were grouped based on effusion volume into one of four groups: full effusions, partial effusions, ears clear of effusion at the time of surgery, and normal control ears. Standard tympanometry was also completed on all ears. RESULTS: Absorbance is systematically reduced as the volume of the middle ear effusion increases. This reduction is present at most frequencies but is greatest in the frequency range from 1 to 5 kHz. A multivariate logistic regression approach was utilized to classify ears based on effusion volume. The regression approach classified ears as effusion present (full and partial ears) or absent (clear ears and normal control ears) with 100% accuracy, ears with effusion present as either partial or full with 100% accuracy, and ears without effusion as either normal control ears or ears clear of effusion with 75% accuracy. Regression performance was also explored when the dataset was split into a training set (70% of the data) and a validation test set (30% of the data) to simulate how this approach would perform on unseen data in a clinical setting. Accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve are reported. Overall, this approach demonstrates high sensitivity and specificity for classifying ears as effusion being present or absent and as present effusions being full or partial with areas under the curve ranging from 1 to 0.944. Despite the lack of effusion present in both clear ears and normal control ears, this approach was able to distinguish between these ears, but with a more moderate sensitivity and specificity. No systematic effect of effusion volume was found on standard tympanometry. CONCLUSIONS: Wideband acoustic immittance, and more specifically, absorbance, is a strong and sensitive indicator of the volume of a middle ear effusion in children with otitis media with effusion.

Invariant asymmetry renews the lymphatic vasculature during homeostasis.

BACKGROUND: The lymphatic vasculature regulates tissue physiology and immunity throughout life. The self renewal mechanism that maintains the lymphatic vasculature during conditions of homeostasis is unknown. The purpose of this study was to investigate the cellular mechanism of lymphatic endothelial cell (LEC) self renewal and lymphatic vessel maintenance. METHODS: Inductive genetic techniques were used to label LECs with tandem dimer tomato (tdT) in adult mice. Two types of studies were performed, those with high dose inductive conditions to label nearly all the lymphatic vessels and studies with low dose inductive conditions to stochastically label individual clones or small populations of LECs. We coupled image guidance techniques and live fluorescence microscopy imaging with lineage tracing to track the fate of entire tdT(+) cutaneous lymphatic vessels or the behavior of individual or small populations of LECs over 11 months. We tracked the fate of 110 LEC clones and 80 small LEC populations (clusters of 2-7 cells) over 11 months and analyzed their behavior using quantitative techniques. RESULTS: The results of the high dose inductive studies showed that the lymphatic vessels remained tdT(+) over 11 months, suggesting passage and expression of the tdT transgene from LEC precursors to progenies, an intrinsic model of self- renewal. Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period. By following the behavior of labeled LEC clones or small populations of LECs individually over 11 months, we identified diverse LEC fates of proliferation, quiescence, and extinction. Quantitative analysis of this data revealed that the average lymphatic endothelial clone or small population remained stable in size despite diverse individual fates. CONCLUSION: The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis. These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis.

Postnatal inflammatory lymphangiogenesis presumably requires precise regulatory processes to properly assemble proliferating lymphatic endothelial cells (LECs). The specific mechanisms that regulate the assembly of LECs during new lymphatic vessel synthesis are unclear. Dynamic endothelial shuffling and rearrangement has been proposed as a mechanism of blood vessel growth. We developed genetic lineage-tracing strategies using an inductive transgenic technology to track the fate of entire tandem dimer tomato-positive (tdT) lymphatic vessels or small, in some cases clonal, populations of LECs. We coupled this platform with a suture-induced mouse model of corneal lymphangiogenesis and used different analytic microscopy techniques including serial live imaging to study the spatial properties of proliferating tdT(+) LEC progenies. LEC precursors and their progeny expanded from the corneal limbal lymphatic vessel and were assembled contiguously to comprise a subunit within a new lymphatic vessel. VE-cadherin blockade induced morphologic abnormalities in newly synthesized lymphatic vessels, but did not disrupt the tdT(+) lymphatic endothelial lineage assembly. Analysis of this static and dynamic data based largely on direct in vivo observations supports a model of lymphatic endothelial lineage assemblage during corneal inflammatory lymphangiogenesis.

Nerve growth factor regulates neurolymphatic remodeling during corneal inflammation and resolution.

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation-pain and swelling-by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.

Lymphatic vessel memory stimulated by recurrent inflammation.

Inflammation stimulates new lymphatic vessel growth (inflammatory lymphangiogenesis). One key question is how recurrent inflammation, a common clinical condition, regulates lymphatic vessel remodeling. We show here that recurrent inflammation accelerated the development a functional lymphatic vessel network. This observation suggests a novel program of lymphangiogenesis and identifies a property of lymphatic vessel memory in response to recurrent inflammation. A brief episode of initial inflammation regressed lymphatic vessels, and a significant increase in CD11b(+) macrophages were associated with the development of lymphatic vessel memory. These vessels had major differences in the structure and the spatial distribution of specialized lymphatic vessel features. Surprisingly, we found that the lymphatic vessel memory response did not depend on the vascular endothelial growth factor C or A pathway, indicating that different molecular pathways regulate inflammatory lymphangiogenesis and lymphatic vessel memory. These findings uncover a priming mechanism to facilitate a rapid lymphatic vessel memory response: a potential important component of peripheral host defense.

Glucocorticoids suppress corneal lymphangiogenesis.

PURPOSE: To determine whether glucocorticoids suppress corneal lymphatic vessel growth (lymphangiogenesis) or induce lymphatic vessel regression. METHODS: We measured human lymphatic endothelial cell proliferation and collagen-induced tubulogenesis in culture conditions with and without dexamethasone, a potent glucocorticoid. We developed a modification of the mouse corneal suture model that allowed us to visualize lymphatic vessel growth (with suture) or regression (suture removed) using immunofluorescence and microscopic techniques. We administered dexamethasone or vehicle control to mice with sutured corneas. We visualized and quantified the corneal lymphatic vessels. We measured vascular endothelial growth factor-C and tumor necrosis factor-α messenger RNA expression in unsutured or sutured corneas using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: High-dose dexamethasone did not change the proliferation or tubulogenesis properties of human lymphatic endothelial cells in vitro. We demonstrated suppressed corneal lymphatic vessel growth rather than lymphatic vessel regression in mice treated with dexamethasone. Expressions of corneal vascular endothelial growth factor-C and tumor necrosis factor-α messenger RNA were similar in mice treated with or without dexamethasone. CONCLUSIONS: Dexamethasone suppressed new lymphatic vessel growth and did not induce lymphatic vessel regression. These findings identify a novel mechanism of glucocorticoid action: suppression of corneal lymphangiogenesis.

Regressed lymphatic vessels develop during corneal repair.

The fate of newly synthesized lymphatic vessels induced by inflammation is poorly understood. To address this question, we designed experiments to determine the morphologic, phenotypic, and functional differences in regressing lymphatic vessels in the context of corneal recovery after an inflammatory response. A suture removal modification was used to induce corneal recovery after suture induced inflammation. We identified an increase in markers of corneal inflammation in sutured cornea that resolved in 14 days after suture removal. Sprouting newly synthesized lymphatic vessels trafficking MHC-II-positive leukocytes were visualized in sutured cornea. Following suture removal and recovery, the visualized lymphatic vessels were thin and fragmented, had bulbous termini, discontinuous expression of CD31 and VE-cadherin, and excluded MHC-II-positive leukocytes. VEGF-A, VEGF-C, and TGF-ß mRNA levels were increased during corneal recovery, suggesting a complex interaction between lymphangiogenic factors and the mechanisms that regulate corneal recovery. The balance of lymphatic vessel growth and regression is likely to have a central role in the pathogenesis of corneal inflammatory diseases.

ß1 integrin regulates MMP-10 dependant tubulogenesis in human lymphatic endothelial cells.

Lymphatic vessel growth requires extensive remodeling of the extracellular matrix, a process hypothesized to be related to the expression and function of the matrix metalloproteinases. We used a protein based screening strategy to demonstrate increased matrix matalloproteinase-10 expression in human lymphatic endothelial cells undergoing collagen I induced tubulogenesis. Knock-down experiments showed that matrix metalloproteinase-10 regulated lymphatic endothelial cell tubulogenesis. ß1 integrin signaling via the ERK/MAPK pathway increased matrix metalloproteinase-10 mRNA and protein expression in human lymphatic endothelial cells. These findings demonstrate a novel mechanism by which ß1 integrin regulates matrix metalloproteinase-10 expression during lymphatic vessel remodeling.

Lymphatic vessel hypertrophy in inflamed human tonsils.

The structural and molecular properties of the human tonsil lymphatic microvascular system are important to understand as these features likely contribute to fluid balance, immunity, and tumor metastasis. The tonsil is a unique lymphoid organ in that it is in intimate contact with the contents of the upper aerodigestive tract and that there are no identifiable afferent lymphatics. Conventional immunofluorescence microscopy demonstrated a remarkable degree of lymphatic vessel architecture within the tonsil; LYVE-1-positive lymphatic vessels were detected around each germinal center and in the marginal regions between the follicles. High resolution confocal laser scanning immunofluoresence microscopy demonstrated that individual lymphatic endothelial cells had a classic 'oak leaf' shape and discontinuous expression of CD31 and VE-cadherin; characteristics hypothesized to be related to fluid and cellular transport. A comparative analysis demonstrated a dramatic increase in lymphatic but not blood vessel density and complexity in inflamed compared to noninflamed tonsil tissue. The results of this study describe the spatial organization of the tonsil lymphatic vasculature, discontinuous expression of CD31 and VE-cadherin in human lymphatic capillaries, and a change in lymphatic vessel morphology in response to inflammation.

Lymphatic malformations: current cellular and clinical investigations.

OBJECTIVE: Summarize current knowledge of lymphatic malformation development, biology, and clinical outcome measures. METHODS: Panel presentation of lymphatic malformation biology and measurement of head and neck malformation treatment outcomes. RESULTS: Characterization of lymphatic malformation endothelial and stromal cells may lead to biologically based treatment. Traditionally, lymphatic malformation treatment outcomes have been measured according to reduction of malformation size. Currently, methods to measure functional outcomes following lymphatic malformation treatment are lacking. This is particularly apparent when the malformation directly involves the upper aerodigestive tract. CONCLUSIONS: The etiology and pathogenesis of head and neck lymphatic malformations are poorly understood, but understanding is improving through ongoing investigation. Reduction of lymphatic malformation size is generally possible, but further work is necessary to optimize methods for measuring therapeutic outcomes in problematic areas.

Lymphatic malformations: review of current treatment.

OBJECTIVE: Summarize current knowledge of lymphatic malformation medical, sclerotherapy, and surgical treatment; and highlight areas of treatment controversy and treatment difficulty that need improvement. METHODS: Panel presentation of various aspects of lymphatic malformation treatment. RESULTS: The mainstay of lymphatic malformation treatment has been surgical resection, which has been refined through lesion staging and radiographic characterization. Intralesional sclerotherapy in macrocystic lymphatic malformations is effective. Suprahyoid microcystic lymphatic malformations are more difficult to treat than macrocystic lymphatic malformations in the infrahyoid and posterior cervical regions. Bilateral suprahyoid lymphatic malformations require staged treatment to prevent complications. Lymphatic malformation treatment planning is primarily determined by the presence or possibility of functional compromise. Problematic areas include chronic lymphatic malformation inflammation, dental health maintenance, macroglossia, airway obstruction, and dental malocclusion. CONCLUSIONS: Lymphatic malformation treatment improvements have been made through radiographic characterization and staging of lymphatic malformations. Direct malformation involvement of the upper aerodigestive tract can cause significant functional compromise that is difficult to treat.

Integrin alpha1beta1 regulates matrix metalloproteinases via P38 mitogen-activated protein kinase in mesangial cells: implications for Alport syndrome.

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.

Clinical outcomes in lymphocytopenic lymphatic malformation patients.

BACKGROUND: To determine if lymphocytopenia in patients with lymphatic malformation (LM) is associated with rates of infection and poor clinical outcomes. MATERIALS AND METHODS: This is a retrospective case series at a tertiary pediatric hospital, of 21 consecutive patients (11 male and 10 female) undergoing LM treatment. Clinical data (i.e., age, clinical LM stage, presence of tissue hypertrophy, frequency/type of medical therapy, and number of hospitalizations) obtained from LM patients with lymphocytopenia (n = 6) was compared to LM patients without lymphocytopenia (n = 15). RESULTS: The average age at the time of detailed leukocyte analysis was 67 months (Range 1-231). Six patients with lymphocytopenia (below 1500/cm(3)) were compared with 15 without lymphocytopenia (above 1500/cm(3)). All six patients with lymphocytopenia had large bilateral LM and normal neutrophil and platelet counts. The total number of hospital admissions was two times greater in lymphocytopenic patients (mean 8.3) compared to nonlymphocytopenic patients (mean 4.09) Chi square analysis revealed a statistical difference in lymphocytopenic patients. They were more likely to have had central line placement, central line infection, bacteremia, prophylactic antibiotics, admission at birth, infections distant from the lymphatic malformation and a treatment complication compared to nonlymphocytopenic patients. Univariate logistic regression revealed that, independent of LM stage, the use of prophylactic antibiotics, the need for a central line, the occurrence of a line infection, and the hospital admission rate were significantly increased in lymphocytopenic patients. CONCLUSION: Patients with LM-associated lymphocytopenia have increased hospitalization requirements, rate of infection, and receive more intensive antibiotic therapy compared to nonlymphocytopenic LM patients.

Lymphocytopenia in children with lymphatic malformation.

OBJECTIVE: To determine whether an immunologic abnormality exists in patients with lymphatic malformation (LM). DESIGN: Retrospective case series. SETTING: Tertiary care pediatric hospital. PATIENTS: Twenty-one consecutive patients (11 male and 10 female) undergoing LM treatment. INTERVENTIONS: Clinical data (ie, age, clinical LM stage, radiographic appearance, and histologic findings) were correlated with complete blood cell count and detailed lymphocyte differential. Complete blood cell counts and lymphocyte subsets were measured in 21 and 18 patients, respectively. RESULTS: The average age at the time of testing was 67 months (range, 1-231 months). The patients were categorized according to LM stage, including 4 (19%) with stage 1, 4 (19%) with stage 2, 4 (19%) with stage 3, 7 (33%) with stage 4, and 2 (10%) with stage 5 disease. Radiographic LM appearance was macrocystic in 6 patients (29%), mixed macrocystic and microcystic in 8 (38%), and microcystic in 7 (33%). Complete blood cell count data demonstrated lymphocytopenia in 6 patients (29%). The results of the lymphocyte subset tests showed concomitant T-, B-, and natural killer (NK)-cell deficiency in 6 (33%) of 18 patients. All 6 patients with T-cell lymphocytopenia had normal neutrophil and platelet counts. Spearman rank and chi(2) analyses showed that LM stage 4 or 5 and microcystic LM were significantly associated with lymphocytopenia (P = .002 and P = .008, respectively). Histologic analysis did not demonstrate increased lymphocytes in any LM specimens. CONCLUSION: We found T, B, and NK lymphocytopenia in patients with large bilateral or microcystic LM. Although the relationship between lymphocytopenia and infection was not addressed in this study, the recognition of lymphocytopenia in patients with LM may have important clinical and prognostic implications.

Proliferation and differentiation of CD8+ T cells in the absence of IL-2/15 receptor beta-chain expression or STAT5 activation.

Major gains in the efficacy of T cell-based therapies for cancer and infectious diseases could be realized through improved understanding of the signals that control expansion and differentiation of CD8(+) cytolytic T cells. IL-2, IL-15, and the downstream transcription factor STAT5 have all been implicated as important regulators of these processes, yet there are conflicting data regarding their contribution to in vivo T cell responses. We used a murine adoptive T cell transfer model to examine the contribution of IL-2 and IL-15 signaling to the proliferation and differentiation of naive, CD8(+) T cells bearing an OVA-specific TCR transgene (OT-I). OT-I T cells failed to express the high affinity IL-2R (CD25) while proliferating in vivo, irrespective of the mode of Ag delivery. Moreover, OT-I T cells rendered genetically deficient in the shared IL-2/IL-15Rbeta subunit (IL-2Rbeta) demonstrated normal Ag-induced proliferation and cytolytic activity in vivo. Accordingly, activation of STAT5 was not detected in proliferating IL-2Rbeta-deficient OT-I T cells, thus implicating a STAT5-independent cytokine or costimulatory pathway in this process. Even though IL-2 and IL-15 were dispensable for CD8(+) T cell proliferation, systemic infusion of IL-2 nevertheless promoted the expansion of OT-I T cells in vivo. Thus, IL-2 and IL-15 signals are not essential for CD8(+) T cell proliferation or differentiation, but IL-2 can promote supraphysiological expansion when supplied exogenously. These findings challenge current models that place CD8(+) T cell proliferation under the control of STAT5-dependent cytokines and suggest new approaches to the therapeutic manipulation of T cell numbers in vivo.

Molecular requirements for CD8-mediated rejection of a MUC1-expressing pancreatic carcinoma: implications for tumor vaccines.

Previous studies have indicated that different effector cells are required to eliminate MUC1-expressing tumors derived from different organ sites and that different vaccine strategies may be necessary to generate these two different MUC1-specific immune responses. In this study, we characterized molecular components that are required to produce immune responses that eliminate Panc02.MUC1 tumors in vivo by utilizing mice genetically deficient in molecules related to immunity. A parallel study has been reported for a B16.MUC1 tumor model. We confirmed that a CD8(+) effector cell was required to eliminate MUC1-expressing Panc02 tumors, and demonstrated that T cells expressing TCR-alpha/beta and co-stimulation through CD28 and CD40:CD40L interactions played critical roles during the initiation of the anti-Panc02.MUC1 immune response. TCR-alpha/beta(+) cells were required to eliminate Panc02.MUC1 tumors, while TCR-gamma/delta(+) cells played a suppressive non-MUC1-specific role in anti-Panc02 tumor immunity. Type 1 cytokine interferon-gamma (IFN-gamma), but not interleukin-12 (IL-12), was essential for eliminating MUC1-expressing tumors, while neither IL-4 nor IL-10 (type 2 cytokines) were required for tumor rejection. In vitro studies demonstrated that IFN-gamma upregulated MHC class I, but not MHC class II, on Panc02.MUC1 tumor cells. Surprisingly, both perforin and FasL played unique roles during the effector phase of immunity to Panc02.MUC1, while lymphotoxin-alpha, but not TNFR-1, was required for immunity against Panc02.MUC1 tumors. The findings presented here and in parallel studies of B16.MUC1 immunity clearly demonstrate that different effector cells and cytolytic mechanisms are required to eliminate MUC1-expressing tumors derived from different organ sites, and provide insight into the immune components required to eliminate tumors expressing the same antigen but derived from different tissues.

MUC1-specific anti-tumor responses: molecular requirements for CD4-mediated responses.

MUC1 was first defined as a tumor antigen in the late 1980s, yet little is known about the types of immune responses that mediate rejection of MUC1(+) tumors in vivo. MUC1-specific antibodies, T(h) cells and cytotoxic T cells can be detected in patients with different adenocarcinomas, yet these tumors usually progress. Thus, there is a need to better understand the in vivo mechanisms of antigen-specific tumor rejection. To characterize the nature of MUC1-specific immune responses in vivo, rejection of a MUC1-expressing melanoma tumor line (B16.MUC1) was evaluated in mice lacking specific T cell subsets, cytokines, co-stimulatory molecules or molecular effectors of cytolytic pathways. Results demonstrated that rejection of the B16.MUC1 tumor cell line was primarily mediated by CD4(+) T cells, and required Fas ligand, lymphotoxin-alpha, CD40, CD40 ligand and CD28, but not perforin, gammadelta T cells, IL-4, IL-10, IL-12 or tumor necrosis factor receptor-1. Depletion of NK cells demonstrated that NK cells might also contribute to MUC1 immunity in the B16.MUC1 tumor model. These results demonstrated that the immune response generated against MUC1 does not fit the type 1 or 2 model described for many immune responses. Additionally, multiple cytolytic mechanisms are required for B16.MUC1 rejection.