P16INK4A immunohistochemistry for detection of human papilloma virus-associated penile squamous cell carcinoma is superior to in-situ hybridization.

We evaluated p16INK4A as a reliable option to detect human papilloma virus (HPV) DNA in penile tumor specimens. Formalin-fixed paraffin embedded samples of 26 patients with penile cancer and another 18 cases with non-tumorigenic lesions were stained by three different widely used commercially available chromogenic in-situ hybridization assays high-risk HPV CISH Y1443 (Genpoint, DAKO), pan HPV CISH Y1404 (Genpoint, DAKO), INFORM HPV III (Ventana, Tucson, Arizona) and p16INK4A immunohistochemistry, then compared to the known gold standard polymerase chain reaction detecting HPV 16, 18, 31, and 33. Immunoreactivity for p16INK4A was evaluated by using a 4-tiered (0, 1, 2, and 3) pattern based system. 19 cases were positive for p16INK4A, 13 of which showed a continuous transepithelial staining (pattern 3). Pan HPV ISH showed positivity in 9 cases, high-risk HPV ISH in 7 cases and INFORM HPVIII ISH in 7 cases. p16INK4A IHC pattern 3 versus pattern 0, 1 and 2 exhibited a specificity and positive predictive value of 100 percent, with a sensitivity and negative predictive value of 72 and 62 percent, respectively, which was much better than all HPV in-situ hybridization methods referred to polymerase chain reaction. p16INK4A seems to be a superior marker for the detection of HPV-associated penile squamous cell carcinoma compared to CISH tests, but is not recommend for the detection of non-tumorigenic lesions, where PCR should be used for the initial assessment.

Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab.

BACKGROUND: Patients with metastatic breast cancer (MBC) overexpressing HER2 (human epidermal growth factor receptor 2) are currently selected for treatment with trastuzumab, but not all patients respond. PATIENTS AND METHODS: Using a novel assay, HER2 protein expression (H2T) was measured in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for MBC. Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T < 13.8), H2T high (H2T ≥ 68.5), and H2T intermediate (13.8 ≤ H2T < 68.5) subgroups. Kaplan-Meier (KM) analyses were carried out comparing the groups for time to progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results. RESULTS: TTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors [median TTP 4.2 months, hazard ratio (HR) = 3.7, P < 0.0001] or H2T high tumors (median TTP 4.6 months, HR = 2.7, P = 0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP 12 months). OS analyses yielded similar results. CONCLUSIONS: MBC patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab. These results are preliminary and require confirmation in larger controlled clinical cohorts.

The impact of lymph node density on survival of cervical cancer patients.

BACKGROUND: To evaluate the prognostic value of lymph node density (LND) in patients with lymph node-positive cervical cancer. METHODS: A total of 88 consecutive patients were included in our study. Patients were treated with cisplatin-based concomitant chemoradiotherapy after surgical staging was performed at the Medical University of Vienna. Lymph node density, that is, the ratio of positive lymph nodes to the total number of lymph nodes removed, was assessed pathologically. Patients were stratified into two groups according to LND: patients with LND 10%. Lymph node density was correlated with clinicopathological parameters by chi(2)-tests. Univariate log-rank tests and multivariate Cox regression models were used to evaluate the association between LND and survival. RESULTS: A significant correlation between LND and FIGO stage (P=0.03), but not patients' age (P=0.2), histological grade (P=0.8), and histological type (P=0.5), was observed. In a univariate survival analysis, LND (P=0.01; P=0.01), FIGO stage (P=0.01; P=0.008), and histological grade (P=0.03; P=0.04) were associated with disease-free and overall survival, respectively. Patients with LND >10% had impaired disease-free and overall survival rates compared with patients with LND 10% is associated with an impaired disease-free and overall survival. Lymph node density may be used as an independent prognostic parameter in patients with lymph node-positive cervical cancer.

Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium.

BACKGROUND: Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c-kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium. METHODS: Immune function of viable peripheral blood mononuclear cells (PBMC) was evaluated after co-culture with irradiated apoptotic PBMC (IA-PBMC) in vitro. Viable PBMC, IA-PBMC and culture supernatants (SN) thereof were obtained after 24 h. Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were utilized to quantify interleukin-8 (IL-8), vascular endothelial growth factor, matrix metalloproteinase-9 (MMP9) in PBMC, SN and SN exposed fibroblasts. Cell suspensions of viable- and IA-PBMC were infused in an experimental rat AMI model. Immunohistological analysis was performed to detect inflammatory and pro-angiogenic cells within 72 h post-infarction. Functional data and determination of infarction size were quantified by echocardiography and Elastica van Gieson staining. RESULTS: The IA-PBMC attenuated immune reactivity and resulted in secretion of pro-angiogenic IL-8 and MMP9 in vitro. Fibroblasts exposed to viable and IA-PBMC derived SN caused RNA increment of IL-8 and MMP9. AMI rats that were infused with IA-PBMC cell suspension evidenced enhanced homing of endothelial progenitor cells within 72 h as compared to control (medium alone, viable-PBMC). Echocardiography showed a significant reduction in infarction size and improvement in post AMI remodelling as evidenced by an attenuated loss of ejection fraction. CONCLUSION: These data indicate that infusion of IA-PBMC cell suspension in experimental AMI circumvented inflammation, caused preferential homing of regenerative EPC and replaced infarcted myocardium.

Myocardial lipofuscin-laden lysosomes contain the apoptosis marker caspase-cleaved cytokeratin-18.

BACKGROUND: Acute coronary syndrome is related to increased circulatory concentration of soluble apoptosis specific caspase-cleaved cytokeratin-18 (ccCK-18). Potential cardiac sources of this intermediate filament derivative have not been investigated to date. MATERIALS AND METHODS: Paraffin embedded tissue of normal myocardium, and chronically damaged samples of ischaemic, congestive and hypertrophic cardiomyopathy were analysed by histology and by CK-8, CK-18, ccCK-18 immunohistochemistry (each group, n = 15). Antibody specificity of the ccCK-18 antibody M30 was checked by immunoblotting on lysed myocardium and enriched myocardial lysosomes. RESULTS: ccCK-18 and CK-18 but not CK-8 were present in all forms of cardiomyopathy, most prominently in ischaemic cardiomyopathy while only traces were detectable immunohistochemically in normal myocardium. Weak CK-18 and strong ccCK-18 staining co-localized to lysosomes with cardiac age pigment lipofuscin. Weak staining of CK-18 was detected in the cytoplasm of coronary endothelia. CONCLUSION: Our study reveals that cardiac lipofuscin-laden lysosomes contain ccCK-18, a marker of apoptosis and its precursor CK-18. This ccCK-18 pool might contribute to increased systemic levels of ccCK-18 in acute coronary syndrome thus monitoring myocardial damage.

Prediction of non-sentinel node status and outcome in sentinel node-positive melanoma patients.

AIMS: Sentinel lymph node (SLN) -positive melanoma patients are usually recommended completion lymph node dissection (CLND) with the aim to provide regional disease control and improve survival. Nevertheless, only 20% these patients have additional metastases in non-sentinel lymph nodes (NSLN), indicating that CLND may be unnecessary in the majority of patients. In this retrospective study, we (i) sought to identify clinico-pathological features predicting NSLN status, as well as disease-free (DFS) and -specific (DSS) survival and (ii) evaluated the applicability of previously published algorithms, which were able to define a group of patients at zero-risk for NSLN-metastasis. METHODS: This analysis included 504 consecutive melanoma patients stage I and II who underwent successful SLN-biopsy (SLNB) at our institute between 1998 and 2005. Metastatic SLN were re-evaluated for tumor burden and categorized according to two different micro-anatomic classifications and the S/U-score (Size of the sentinel node metastasis > 2 mm/Ulceration of the primary melanoma) was assessed. DFS and DSS were calculated for all analyses. RESULTS: Out of 504 melanoma patients stage I or II, 85 (17%) were SLN-positive and 18 of 85 (21%) were found with positive NSLN in the CLND specimen. Median follow-up was 31 months. Neither primary tumor characteristics (age, gender, Clark level, Breslow thickness, ulceration of the primary melanoma, site and histological subtype of the primary melanoma), nor features of the sentinel node tumor (number and site of draining lymph node basins, number of positive sentinel nodes and size of sentinel node tumor (< 2 mm vs. > or = 2 mm) were able to predict additional positive lymph nodes in the CLND specimen. Likewise the implementation of published algorithms was not able to identify patients at negligible risk for harboring NSLN metastases. Upon univariate analysis, disease-free survival in SLN-positive patients was correlated with Breslow thickness, sentinel node tumor size > 2 mm and S/U score. In respect to disease-specific survival the significant prognostic parameters were Breslow thickness, ulceration, sentinel node tumor size > 2 mm and the S/U score. After a median follow-up of 31 months recurrence rates (37% vs. 78%, p=0.02) and death from disease (24% vs. 50%, p<0.01) were significantly different in patients with SLN-metastasis only as compared to patients with NSLN-metastasis. CONCLUSION: NSLN status cannot be predicted in this data analysis by using clinico-pathological characteristics. Therefore, CLND is recommended for all patients after positive SLNB pending the results of the second Multicenter Selective Lymphadenectomy Trial.

Plaque imaging of the internal carotid artery - correlation of B-flow imaging with histopathology.

BACKGROUND AND PURPOSE: The noninvasive identification of plaque types prone to cause symptomatic disease is of great interest to improve the effectiveness of surgical or interventional management. The purpose of the present prospective pilot study was to evaluate the association between the results of imaging-the novel sonography technique B-flow imaging (BFI), B-mode, and color Doppler imaging (CDI)-and histopathologic examination in the characterization of internal carotid artery (ICA) plaques. METHODS: Twenty-eight consecutive patients with high-grade internal carotid artery stenosis scheduled for carotid endarterectomy were included. BFI, B-mode, and CDI images were used to classify the plaques applying the standardized scores of Beletsky et al and the American Heart Association (AHA), to calculate the gray-scale median (GSM) and to detect potential ulcerations; histopathologic examination results of explanted plaques served as the "gold standard." RESULTS: Based on the classification of Beletsky et al, BFI and histopathologic examination results agreed in 21 (75%, kappa = 0.61, P < .001) patients, and the corresponding results for B-mode were 19 (68%, kappa = 0.52, P < .001) patients, respectively. Corresponding results for the AHA classification revealed inferior agreements for BFI (19 patients/68%, kappa = 0.38, P = .003) and B-mode (17 patients/61%, kappa = 0.25, P = .045). The median GSM for BFI and B-mode correlated significantly (r = 0.95, P < .001). The sensitivity of BFI for the detection of ulcerated plaques was 100% and the specificity was 95.8%; corresponding values for CDI were 100% and 92.7%, respectively. CONCLUSION: BFI and the combination of B-mode and CDI exhibit comparable results in the assessment of ICA plaque components and plaque ulceration as well as in the determination of GSM levels.

In ovarian cancer the prognostic influence of HER2/neu is not dependent on the CXCR4/SDF-1 signalling pathway.

HER2/neu overexpression is a driving force in the carcinogenesis of several human cancers. In breast cancer the prognostic influence of HER2/neu was shown to be at least partly based on increased metastatic potential mediated by the chemokine-chemokine receptor pair SDF-1(CXCL12)/CXCR4. We wanted to evaluate the influence of HER2/neu on ovarian cancer prognosis and to investigate whether compromised survival would correlate with CXCR4 expression and/or SDF-1 abundance. Therefore, we analysed HER2/neu, CXCR4, and SDF-1 in 148 ovarian tumour samples by means of immunohistochemistry on tissue microarrays. Overexpression of HER2/neu was found in 27.6% of ovarian cancer tissues and in 15% of ovarian borderline tumours. In ovarian cancer patients, overexpression of HER2/neu correlated closely with overall survival (univariate hazard ratio (HR) 2.59, P=0.005; multiple corrected HR 1.92, P=0.074). In contrast, CXCR4 expression and SDF-1 abundance had no impact on overall survival, and both parameters were not correlated with HER2/neu expression. As expected, cytoplasmic CXCR4 expression and SDF-1 abundance correlated closely (P<0.0001). Our results confirm a univariate influence of HER2/neu expression on overall survival, which was completely independent of the expression of CXCR4 and the abundance of SDF-1, implying significant differences between the HER2/neu downstream pathways in ovarian cancer compared with breast cancer.

Most of diaplacentally transferred allergen is retained in the placenta.

BACKGROUND: Transplacental transfer of nutritive and inhalant allergens has been described being potentially responsible for a series of events leading to antigen-specific immune responses in the fetus. As such, cord blood T cell responses appear ubiquitously. However, studies failed to reveal a consistent dose-response relationship between antenatal allergen exposure and allergen-specific cellular reactivity in cord blood. OBJECTIVE: To examine the transfer process of allergens (ovalbumin (OVA), beta-lactoglobulin (BLG), birch pollen allergen Bet v1) in placental tissue (BeWo cell line, ex vivo placenta model). METHODS: The choriocarcinoma cell line BeWo was used to study the allergen uptake and transfer experiments in vitro. In the ex vivo placenta model the contribution of different placental compartments was evaluated. For this, immuno-histochemistry, immuno-electronmicroscopy and ELISA techniques were applied using monoclonal antibodies to Bet v1, OVA and -BLG. RESULTS: In vitro transfer studies on a BeWo cell-layer revealed an intracellular allergen uptake and a trans-trophoblastic allergen transfer, which was temperature- and concentration dependent, pH sensitive and asymmetric. Allergen-specific staining of placental tissue after allergen perfusion (BLG) demonstrated bulk of the allergen in the syncytio-trophoblastic cell layer and minor staining in the villous stroma and in the endothelium of fetal vessels. Immunogold staining revealed an accumulation of the perfused allergen in the trophoblastic basement membrane. CONCLUSION: In vitro/ex vivo trans-trophoblastic and trans-placental allergen transfer is shown with an accumulation of most of the allergen in placental tissues, potentially explaining the missing direct dose-response relationship between prenatal (maternal) allergen exposure and allergen-specific cellular reactivity in cord blood.

Recovery from giant cell myocarditis with ECMO support and utilisation of polyclonal antithymocyte globulin: a case report.

Giant cell myocarditis (GCM) is a rare and frequently fatal disorder. Patients suffer of ventricular arrhythmias or congestive heart failure. Here we describe a patient with cardiogenic shock and histological verified GCM. The patient was saved by implantation of extracorporeal membrane oxygeneation (ECMO) device and concomitant application of Rabbit antithymocyte globuline (rATG, Thymoglobulin, Sangstat), cyclosporine, and steroids in the acute event. 12 months after the crisis the patient evidences NYHA class I heart function and only a moderate impairment of heart function (EF 55%). The novel utilisation of ECMO in GCM related cardiogenic shock and application of rATG have prooven life-saving in this patient. Studies utilizing rATG in the treatment of GCM are warrented.

Oct-4 expression in human endometrium.

The transcription factor Oct-4 is crucial for the maintenance of cell pluripotency and is known to be expressed in embryonic stem cells, germ cells and whole embryos at various stages of development. Oct-4 regulates cell fate in a dose-dependent manner and plays a key role in germ-cell tumours. In the past, several stem-cell markers have been detected, and their role in the pathogenesis of diseases has been discussed frequently. Thus, we investigated the expression of Oct-4 comparing its occurrence in endometrium of healthy and diseased women using immunohistochemistry (IHC) and RT-PCR. IHC demonstrated Oct-4 expression in 25 of 60 sections (42%), respectively in 11 out of 25 patients (44%). Oct-4 mRNA was detected by RT-PCR in all tested samples (9 of 9) of endometrium, although the levels of expression varied. To our knowledge, this is the first study demonstrating Oct-4 expression in human endometrium.

Expression of RPE65, a putative receptor for plasma retinol-binding protein, in nonmelanocytic skin tumours.

BACKGROUND: In a recent report we described RPE65, a protein originally characterized in retinal pigment epithelium, to be expressed in normal human epidermis. RPE65 is suspected to be involved in cellular uptake of retinol which is transported in the bloodstream complexed with plasma retinol-binding protein. OBJECTIVES: To evaluate protein and mRNA expression of RPE65 in actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) compared with normal skin. METHODS: RPE65 mRNA expression in skin tumours relative to normal skin of the respective donor was studied by real-time polymerase chain reaction in AK (n = 15), invasive SCC (n = 30) and BCC (n = 18). A peptide-specific anti-RPE65 antibody was used for immunohistochemical staining of formalin-fixed and paraffin-embedded tissue sections of the respective tumours. RESULTS: RPE65 mRNA expression was reduced in AK. A highly significant reduction of RPE65 mRNA was observed in invasive SCC relative to normal skin of the respective donors. Immunohistochemistry revealed a continuous staining of basal and suprabasal keratinocytes in normal human epidermis. RPE65 in AK shown by immunohistochemical staining was reduced and quite irregular, whereas invasive SCC revealed no staining of tumour cells with the anti-RPE65 antibody. RPE65 mRNA values were elevated, whereas immunohistochemical staining for RPE65 protein was heterogeneous in BCC. CONCLUSIONS: These results suggest progressive downregulation of RPE65 from AK to invasive SCC.

Semicircular tumor of the iris and uveitis as unilocal manifestation of sarcoidosis.

PURPOSE: To describe an unusual manifestation of sarcoidosis as a large tumor of the iris and ciliary body without any other involvement of the body. METHODS: We describe a 20-year-old female presenting with a granulomatous tumor of the right iris and ciliary body and concomitant uveitis. RESULTS: Extensive ocular and systemic workup revealed the tumor to be a large solitary sarcoid granuloma. As systemic steroids were not able to control the activity of the uveitis and granuloma, only the initiation of immunosuppressive therapy with cyclosporine A achieved a lasting remission. CONCLUSION: The possibility of an exclusively ocular sarcoidosis should always be kept in mind despite negative regular screening tests. In these cases, a biopsy should be considered and immunosuppressive agents like cyclosporine A should be evaluated in cases not responding to first-line treatment with systemic steroids.

Cathepsin D in ovarian cancer: prognostic value and correlation with p53 expression and microvessel density.

OBJECTIVE: Overexpression of ubiquitous lysosomal aspartyl protease cathepsin D (CD) is involved in the progression of cancer. This study investigates the prognostic value and the association of cathepsin D expression with clinicopathological parameters, p53 expression, and angiogenesis in ovarian cancer. METHODS: Cathepsin D was determined immunohistochemically in 43 ovarian tumors of low malignant potential (LMP) and 80 invasive tumors FIGO stage I-IV. Results were correlated with clinicopathological characteristics, p53, and microvessel density (MVD). Survival analysis of cathepsin D expression and MVD was performed in invasive tumors. RESULTS: Epithelial tumor cathepsin D expression was more common in LMP tumors (65.1%) compared to invasive tumors (43.7%; P = 0.02). In LMP tumors, stromal cathepsin D was associated with mucinous tumors (P = 0.01), whereas in invasive tumors, epithelial cathepsin D expression was associated with clear cell tumors (P = 0.003). Invasive tumor cathepsin D had a negative relation to p53 expression. In LMP tumors, stromal cathepsin D correlated with microvessel density (P = 0.03). Stromal cathepsin D expression was an independent prognostic factor for disease-free survival (DFS) in patients with invasive cancer (P = 0.03, Cox regression), while cathepsin D expression missed to be of prognostic value for overall survival (OS) in invasive ovarian cancer. MVD had no influence on survival in invasive ovarian cancer (P > 0.05). CONCLUSION: Our study demonstrates a prognostic value of cathepsin D expression in invasive ovarian cancer, while cathepsin D expression in LMP tumors seems to be linked to angiogenesis. The relation among cathepsin D, p53 expression, and angiogenesis demonstrates biological differences between invasive ovarian cancer and LMP tumors.

Biofilm detection and the clinical significance of Staphylococcus epidermidis isolates.

The ability of Staphylococcus epidermidis to produce biofilm was compared in 147 clinically significant strains repeatedly isolated from blood cultures of patients with bloodstream infection and in 147 strains isolated from skin. The strains were examined for the presence of ica operone, for the ability to form biofilm by Christensen's test-tube method and for the production of slime by Congo Red agar method. The ica operone was found in 92 (62.6 %) blood isolates and in 44 (29.9) isolates from skin. Christensen's test-tube method was positive in 79 (53.7) and 33 (22.4), Congo Red agar method in 64 (43.5) and 31 (21.1) of blood and skin isolates, respectively. All three methods were more frequently positive in clinically significant isolates from blood than in strains isolated from skin. The detection of ica operone and the Christensen's test-tube method showed better correlation with the clinical significance than the Congo Red agar method.

Immunohistochemical expression of laminin-5 in cervical intraepithelial neoplasia.

OBJECTIVES: Laminin-5 is an attachment protein for epithelial cells. Several studies of a variety of cancers have reported increased expression of laminin-5 in carcinoma in situ and invasive cancer. This study was designed to investigate the correlation between the grade of cervical intraepithelial neoplasia and the immunohistochemical expression of laminin-5 in the cytoplasm and in the basement membrane underlining dysplastic squamous cells. METHODS: We used immunohistochemical methods to stain paraffin-embedded sections of cervical cone biopsies with a monoclonal antibody specifically targeting the 2-chain of human laminin-5 protein. The study sample included 175 slides: 7 normal cervical epithelium, 36 lesions of mild dysplasia, 50 lesions of moderate dysplasia, 81 lesions of severe dysplasia, and 1 invasive squamous cell carcinoma. RESULTS: We found a statistically significant correlation between the grade of cervical intraepithelial neoplasia and laminin-5 immunoreactivity in the cytoplasm (P < 0.01) and in the basement membrane (P = 0.03) by use of the Wilcoxon rank-sum test. CONCLUSIONS: According to previously published reports we confirmed with a higher number of cases a correlation of laminin-5 expression in the cytoplasm and/or basement membrane and grade of dysplastic lesion in the cervical epithelium. This study warrants further investigations with special interest to follow-up to investigate whether laminin-5 is a marker to predict the risk of progression of cervical intraepithelial neoplasia lesions.

Molecular dynamics of point mutated I-A(k) molecules expressed on lymphocytes.

We have recently reported the lateral and rotational diffusion parameters for I-A(k) molecules expressing various cytoplasmic truncations (Int. Immunol. 12 (2000) 1319). We now describe the membrane dynamics of I-A(k) with various mutations in the presumed contact region between alphabeta-heterodimers in an (alphabeta)2 dimer of dimers structure. Such mutations are known to strongly affect the antigen presentation ability of these molecules (Int. Immunol. 10 (1998) 1237-1249) but cause relatively small changes in the molecular dynamics of I-A(k). Lateral diffusion coefficients of I-A(k) wild-type molecules and mutants obtained via fringe fluorescence photobleaching recovery (FPR) ranged from 1.1 to 2.3x10(-10)cm2/s at room temperature while fractional mobilities averaged 75+/-6%. For all cell types examined, treatment with either hen egg lysozyme 46-61 peptide or db-cAMP reduced the I-A(k) mobile fraction by about 10% relative to untreated cells, suggesting that these treatments may increase lateral confinement of class II in lipid rafts or cytoskeletal interactions of the molecules. Wild-type I-A(k) and mutants capable of normal or partial antigen presentation exhibited, as a group, slightly longer rotational correlation times (RCT) at 4 degrees C than did mutants inactive in antigen presentation, 14+/-4 versus 10+/-1 micros, respectively. Moreover, peptide, cAMP and anti-CD40 mAb treatment all increased rotational correlation times for fully- and partially-functional I-A(k) but not for non-functional molecules. For example, 16 h peptide treatment yielded average RCTs of 28+/-12 and 10+/-1 micros for the groups of functional and non-functional molecules, respectively. Such modulation of the dynamics of functional class II molecules is consistent with these treatments' stabilization of class II or induction of new gene expression. Measurements of fluorescence resonant energy transfer between I-A(k), though complicated by cellular autofluorescence, averaged 6+/-7% over 15 cells or treatments, a result consistent with the presence of a small fraction of I-A(k) as a dimer of dimers species. In summary, our results suggest subtle changes in the molecular motions of class II molecules correlate with a significant impact on class II function. Molecules active in antigen presentation exhibit more restricted motion in the membrane, and thus presumably more extensive intermolecular interactions, than non-functional molecules. Further, treatments, such as db-cAMP and anti-CD40, which rescue antigen presentation by partially defective mutants, appear to increase such interactions, several types of which have already been reported for class II. A more detailed understanding of these phenomena will require both more sensitive biophysical tools and a more refined model of the role of class II intermolecular interactions in antigen presentation.