Effusion-synovitis worsening mediates the association between body mass index and Kellgren-Lawrence progression in obese individuals: data from the Osteoarthritis Initiative.

OBJECTIVE: Both obesity and synovitis are independently associated with knee osteoarthritis (KOA) progression. We examined whether synovitis mediates the relationship between body mass index (BMI) and KOA radiographic progression in the Osteoarthritis Initiative (OAI) cohort. DESIGN: We conducted a case-control study within the OAI. Cases (n = 315) were right knees with an increase of ≥1 Kellgren-Lawrence from baseline to 48 months of follow-up. Controls (n = 315) were right knees with no KL change. Cases and controls were matched by age, sex, race, and baseline KL. MRI Osteoarthritis Knee Score (MOAKS) at baseline and at 2 years was used for a semi-quantitative scoring (0-3) of effusion-synovitis and Hoffa-synovitis. Conditional logistic regression estimated associations between BMI and synovitis with KOA progression. Mediation analysis was used to assess the mediating effects of synovitis. RESULTS: The mean age of participants was 61 years, 70.8% were women, and 87% were White. KOA progression was associated with higher BMI (adjusted OR 1.05; 95%CI 1.01-1.09) and effusion-synovitis relative to no effusion-synovitis (adjusted OR 2.2; 95%CI 1.6-3.1). Associations between effusion-synovitis worsening and KOA progression were more pronounced among obese individuals (OR 34.1; 95%CI 4.2-274.8; P = 0.001) compared to normal weight (OR 3.2; 95%CI 0.8-12.8, P=0.096) individuals. Effusion-synovitis at 2 years, but not at baseline, mediated the relationship between BMI and KOA progression over a 4-year period. CONCLUSIONS: We found that effusion-synovitis worsening mediated the association between BMI and KOA progression and was associated with increased risk of KOA progression, particularly among obese individuals.

Macromolecular fraction (MMF) from 3D ultrashort echo time cones magnetization transfer (3D UTE-Cones-MT) imaging predicts meniscal degeneration and knee osteoarthritis.

OBJECTIVE: Meniscal degeneration is strongly associated with osteoarthritis (OA). We aimed to evaluate a 3D ultrashort-echo-time Cones magnetization transfer (UTE-Cones-MT) sequence for quantification of macromolecular fraction (MMF) and MT ratio (MTR) in menisci of healthy volunteers and patients with different degrees of OA. METHODS: Patients with mild OA (n = 19; 37-86 years; 10 males) or advanced OA (n = 12; 52-88 years; 4 males) and healthy volunteers (n = 17; 20-49 years; 7 males) were scanned with T2-FSE and UTE-Cones-MT sequences at 3T. Morphological assessment was performed using meniscal whole-organ magnetic resonance imaging score (WORMS). MMF and MTR were calculated for menisci, and correlated with age and meniscal WORMS scores. The diagnostic efficiency was performed by using receiver operating characteristic (ROC) curve and the area under the curve (AUC) analyses. RESULTS: Decreased MMF and MTR were observed in menisci of patients with mild or advanced OA compared with healthy subjects, and in menisci with tears (Grade 2-4) compared with normal menisci (Grade 0). Significant negative correlations were observed between MMF (r = -0.769, P < 0.01), MTR (r = -0.320, P < 0.01), and meniscal WORMS score. There was a mild negative correlation between MMF (r = -0.438, P < 0.01), MTR (r = -0.289, P < 0.01), and age. The AUC values of MMF and MTR in the four horns of meniscus and the posterior horn medial meniscus for differentiating OA patients from healthy volunteers were 0.762 and 0.699, and 0.835 and 0.883, respectively. CONCLUSION: The 3D UTE-Cones-MT biomarkers of MTR and especially MMF can detect compositional changes in meniscus and differentiate healthy subjects from patients with mild or advanced knee OA.

Inversion Recovery Ultrashort TE MR Imaging of Myelin is Significantly Correlated with Disability in Patients with Multiple Sclerosis.

BACKGROUND AND PURPOSE: MR imaging has been widely used for the noninvasive evaluation of MS. Although clinical MR imaging sequences are highly effective in showing focal macroscopic tissue abnormalities in the brains of patients with MS, they are not specific to myelin and correlate poorly with disability. We investigated direct imaging of myelin using a 2D adiabatic inversion recovery ultrashort TE sequence to determine its value in assessing disability in MS. MATERIALS AND METHODS: The 2D inversion recovery ultrashort TE sequence was evaluated in 14 healthy volunteers and 31 patients with MS. MPRAGE and T2-FLAIR images were acquired for comparison. Advanced Normalization Tools were used to correlate inversion recovery ultrashort TE, MPRAGE, and T2-FLAIR images with disability assessed by the Expanded Disability Status Scale. RESULTS: Weak correlations were observed between normal-appearing white matter volume (R = -0.03, P = .88), lesion load (R = 0.22, P = .24), and age (R = 0.14, P = .44), and disability. The MPRAGE signal in normal-appearing white matter showed a weak correlation with age (R = -0.10, P = .49) and disability (R = -0.19, P = .31). The T2-FLAIR signal in normal-appearing white matter showed a weak correlation with age (R = 0.01, P = .93) and disability (R = 0.13, P = .49). The inversion recovery ultrashort TE signal was significantly negatively correlated with age (R = -0.38, P = .009) and disability (R = -0.44; P = .01). CONCLUSIONS: Direct imaging of myelin correlates with disability in patients with MS better than indirect imaging of long-T2 water in WM using conventional clinical sequences.

Acquired Hemophilia A (FVIII Deficiency) Associated with Papillary Thyroid Cancer: Treatment with Recombinant Porcine FVIII.

Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by autoantibodies against Factor VIII (FVIII). It has a high mortality due to bleeding complications. FVIIa-based bypassing agents are the first line of treatment but not always effective. Recombinant porcine (rp) FVIII (Obizur®) was recently approved for rescue treatment but with little evidence-based information regarding efficacy. We report a case of papillary thyroid cancer associated with AHA malignancy that responded to a single dose of rpFVIII after failure to achieve hemostasis with FVIIa-based bypassing products.

Musculoskeletal ultrasound for intra-articular bleed detection: a highly sensitive imaging modality compared with conventional magnetic resonance imaging.

Essentials The best imaging modality for joint blood detection in hemophilia is unknown. Blood appearance and detection thresholds were studied with ultrasound and conventional MRI. Ultrasound is sensitive to low volume and concentration of blood, whereas conventional MRI is not. The findings establish the validity of ultrasound for rapid bleed detection in hemophilia care. SUMMARY: Background There is increasing demand for musculoskeletal ultrasound (MSKUS) to detect hemophilic joint bleeding, but there is uncertainty regarding blood detection concentration thresholds or if magnetic resonance imaging (MRI) is more accurate. Aims Compare the sensitivity of blood detection by MSKUS and MRI. Methods Increasing blood concentrations in plasma were imaged with MSKUS and MRI 1-2 h, 3-4 days and 7 days after blood withdrawal in vitro, and after injection into cadaveric pig joints. Additionally, effusions in the joints of two patients with hemophilia joints were imaged, followed by aspiration. MSKUS was performed using an 8-18-MHz linear transducer; MRI was performed at 3T using T1-weighted and T2-weighted fat-suppressed sequences. Images were reviewed by a hematologist certified in MSKUS and a musculoskeletal radiologist. Results MSKUS permitted the detection of blood in vitro and in pig joint spaces at concentrations as low as 5%, demonstrated by the presence of echogenic signals that were absent with plasma alone. In contrast, no differences between fluids were discernible on the T1-weighted or T2-weighted MRI images. Results were confirmed in the two patients with hemophilia. Blood clots demonstrated varying and dynamic echogenicity patterns over time and, using MRI, were visualized best with T2 sequences. Conclusion MSKUS is extremely sensitive in detecting low concentrations of intra-articular blood and in discriminating between bloody and non-bloody fluid, whereas conventional MRI is not. These observations demonstrate the advantages of MSKUS over MRI in detecting intra-articular blood, and show that MSKUS is ideal for rapid bleed detection in the clinic.

Magic angle effect plays a major role in both T1rho and T2 relaxation in articular cartilage.

PURPOSE: To investigate the effect of sample orientation on T1rho and T2 values of articular cartilage in histologically confirmed normal and abnormal regions using a whole-body 3T scanner. MATERIALS AND METHODS: Eight human cadaveric patellae were evaluated using a 2D CPMG sequence for T2 measurement as well as a 2D spin-locking prepared spiral sequence and a 3D magnetization-prepared angle-modulated partitioned-k-space spoiled gradient echo snapshots (3D MAPSS) sequence for T1rho measurement. Each sample was imaged at six angles from 0° to 100° relative to the B0 field. T2 and T1rho values were measured for three regions (medial, apex and lateral) with three layers (10% superficial, 60% middle, 30% deep). Multiple histopathologically confirmed normal and abnormal regions were used to evaluate the angular dependence of T2 and T1rho relaxation in articular cartilage. RESULTS: Our study demonstrated a strong magic angle effect for T1rho and T2 relaxation in articular cartilage, especially in the deeper layers of cartilage. On average, T2 values were increased by 231.8% (72.2% for superficial, 237.6% for middle, and 187.9% for deep layers) while T1rho values were increased by 92% (31.7% for superficial, 69% for middle, and 140% for deep layers) near the magic angle. Both normal and abnormal cartilage showed similar T1rho and T2 magic angle effect. CONCLUSIONS: Changes in T1rho and T2 values due to the magic angle effect can be several times more than that caused by degeneration, and this may significantly complicate the clinical application of T1rho and T2 as an early surrogate marker for degeneration.

UTE bi-component analysis of T2* relaxation in articular cartilage.

OBJECTIVES: To determine T2* relaxation in articular cartilage using ultrashort echo time (UTE) imaging and bi-component analysis, with an emphasis on the deep radial and calcified cartilage. METHODS: Ten patellar samples were imaged using two-dimensional (2D) UTE and Car-Purcell-Meiboom-Gill (CPMG) sequences. UTE images were fitted with a bi-component model to calculate T2* and relative fractions. CPMG images were fitted with a single-component model to calculate T2. The high signal line above the subchondral bone was regarded as the deep radial and calcified cartilage. Depth and orientation dependence of T2*, fraction and T2 were analyzed with histopathology and polarized light microscopy (PLM), confirming normal regions of articular cartilage. An interleaved multi-echo UTE acquisition scheme was proposed for in vivo applications (n = 5). RESULTS: The short T2* values remained relatively constant across the cartilage depth while the long T2* values and long T2* fractions tended to increase from subchondral bone to the superficial cartilage. Long T2*s and T2s showed significant magic angle effect for all layers of cartilage from the medial to lateral facets, while the short T2* values and T2* fractions are insensitive to the magic angle effect. The deep radial and calcified cartilage showed a mean short T2* of 0.80 ± 0.05 ms and short T2* fraction of 39.93 ± 3.05% in vitro, and a mean short T2* of 0.93 ± 0.58 ms and short T2* fraction of 35.03 ± 4.09% in vivo. CONCLUSION: UTE bi-component analysis can characterize the short and long T2* values and fractions across the cartilage depth, including the deep radial and calcified cartilage. The short T2* values and T2* fractions are magic angle insensitive.

Control of metamorphic buffer structure and device performance of In(x)Ga(1-x)As epitaxial layers fabricated by metal organic chemical vapor deposition.

Using a step-graded (SG) buffer structure via metal-organic chemical vapor deposition, we demonstrate a high suitability of In0.5Ga0.5As epitaxial layers on a GaAs substrate for electronic device application. Taking advantage of the technique's precise control, we were able to increase the number of SG layers to achieve a fairly low dislocation density (∼10(6) cm(-2)), while keeping each individual SG layer slightly exceeding the critical thickness (∼80 nm) for strain relaxation. This met the demanded but contradictory requirements, and even offered excellent scalability by lowering the whole buffer structure down to 2.3 µm. This scalability overwhelmingly excels the forefront studies. The effects of the SG misfit strain on the crystal quality and surface morphology of In0.5Ga0.5As epitaxial layers were carefully investigated, and were correlated to threading dislocation (TD) blocking mechanisms. From microstructural analyses, TDs can be blocked effectively through self-annihilation reactions, or hindered randomly by misfit dislocation mechanisms. Growth conditions for avoiding phase separation were also explored and identified. The buffer-improved, high-quality In0.5Ga0.5As epitaxial layers enabled a high-performance, metal-oxide-semiconductor capacitor on a GaAs substrate. The devices displayed remarkable capacitance-voltage responses with small frequency dispersion. A promising interface trap density of 3 × 10(12) eV(-1) cm(-2) in a conductance test was also obtained. These electrical performances are competitive to those using lattice-coherent but pricey InGaAs/InP systems.

A comprehensive, multispecialty approach to an acute exacerbation of chronic central pain in a tetraplegic.

STUDY DESIGN: We present a case report describing the multidisciplinary treatment of a tetraplegic spinal cord injury (SCI) patient who developed an acute exacerbation of chronic central pain. OBJECTIVE: To bring further awareness to the importance of using a comprehensive, multidisciplinary approach in treating acute exacerbation of chronic central pain in SCI patients. SETTING: University of California Irvine Medical Center, Orange, CA, USA. CASE REPORT: We present a 34-year-old man with a past medical history of C5 American Spinal Injury Association B tetraplegia secondary to a surfing accident 8 years prior, central pain syndrome, spasticity, autonomic dysreflexia and anxiety who arrived at the emergency room with a 1-month history of worsening acute on chronic pain refractory to opioid escalation. The multispecialty treatment plan included treatment of the patient's urinary tract infection by the primary medicine service, management of the patient's depression by the psychiatric service, treatment of bowel obstruction by general surgery and adjustment of pain medications by pain management. The patient was found to have stable neurological findings, neuroimaging unchanged from prior imaging and a urinary tract infection. Hospitalization was complicated by severe colonic dilation that required disimpaction by general surgery. CONCLUSION: The treatment of this patient's acutely worsened central pain highlights the importance of applying a multidisciplinary approach to SCI patients with an acute exacerbation of chronic central pain. In this case, the multispecialty treatment plan included treatment of the patient's urinary tract infection by the primary medicine service, management of the patient's depression by the psychiatric service, treatment of bowel obstruction by general surgery, and adjustment of pain medications by pain management.

Spinal 5-HT3 receptors facilitate behavioural hypersensitivity induced by elevated calcium channel alpha-2-delta-1 protein.

BACKGROUND: Peripheral nerve injury induces up-regulation of the calcium channel alpha-2-delta-1 proteins in the dorsal root ganglia and dorsal spinal cord that correlates with neuropathic pain development. Similar behavioural hypersensitivity was also observed in injury-free transgenic (TG) mice over-expressing the alpha-2-delta-1 proteins in neuronal tissues. To investigate pathways regulating alpha-2-delta-1 protein-mediated behavioural hypersensitivity, we examined whether spinal serotonergic 5-HT3 receptors are involved similarly in the modulation of behavioural hypersensitivity induced by either peripheral nerve injury in a nerve injury model or neuronal alpha-2-delta-1 over-expression in the TG model. METHODS: The effects of blocking behavioural hypersensitivity in these two models by intrathecal or systemic injections of 5-HT3 receptor antagonist, ondansetron, were compared. RESULTS: Our data indicated that the TG mice displayed similar behavioural hypersensitivities to non-painful mechanical stimulation (tactile allodynia) and painful thermal stimulation (thermal hyperalgesia) as that observed in the nerve injury model. Interestingly, tactile allodynia and thermal hyperalgesia in both models can be blocked similarly by intrathecal, but not systemic, injection of ondansetron. CONCLUSIONS: Our data suggest that spinal 5-HT3 receptors are likely to play a role in alpha-2-delta-1-mediated behavioural hypersensitivities through a descending serotonergic facilitation.

Dual inversion recovery ultrashort echo time (DIR-UTE) imaging and quantification of the zone of calcified cartilage (ZCC).

OBJECTIVE: To develop ultrashort echo time (UTE) magnetic resonance imaging (MRI) techniques to image the zone of calcified cartilage (ZCC), and quantify its T2*, T1 and T1ρ. DESIGN: In this feasibility study a dual inversion recovery UTE (DIR-UTE) sequence was developed for high contrast imaging of the ZCC. T2* of the ZCC was measured with DIR-UTE acquisitions at progressively increasing TEs. T1 of the ZCC was measured with saturation recovery UTE acquisitions at progressively increasing saturation recovery times. T1ρ of the ZCC was measured with spin-locking prepared DIR-UTE acquisitions at progressively increasing spin-locking times. RESULTS: The feasibility of the qualitative and quantitative DIR-UTE techniques was demonstrated on phantoms and in six cadaveric patellae using a clinical 3 T scanner. On average the ZCC has a short T2* ranging from 1.0 to 3.3 ms (mean ± standard deviation = 2.0 ± 1.2 ms), a short T1 ranging from 256 to 389 ms (mean ± standard deviation = 305 ± 45 ms), and a short T1ρ ranging from 2.2 to 4.6 ms (mean ± standard deviation = 3.6 ± 1.2 ms). CONCLUSION: UTE MR based techniques have been developed for high resolution imaging of the ZCC and quantitative evaluation of its T2*, T1 and T1ρ relaxation times, providing non-invasive assessment of collagen orientation and proteoglycan content at the ZCC and the bone cartilage interface. These measurements may be useful for non-invasive assessment of the ZCC, including understanding the involvement of this tissue component in osteoarthritis.

Endoscopic ultrasound for the evaluation of Nissen fundoplication integrity: a blinded comparison with conventional testing.

BACKGROUND: For patients whose symptoms develop after Nissen fundoplication, the precise mechanism of anatomic failure can be difficult to determine. The authors have previously reported the endosonographic hallmarks defining an intact Nissen fundoplication in swine and the known causes of failure. The current clinical trial tested the hypothesis that a defined set of endosonographic criteria can be applied to determine fundoplication integrity in humans. METHODS: The study enrolled seven symptomatic and nine asymptomatic subjects at a mean of 6 years (range, 1-30 years) after Nissen fundoplication. A validated gastroesophageal reflux disease (GERD)-specific questionnaire and medication history were completed. Before endoscopic ultrasound (EUS), all the patients underwent complete conventional testing (upper endoscopy, esophagram, manometry, 24-h pH). A diagnosis was rendered on the basis of combined test results. Then EUS was performed by an observer blinded to symptoms, medication use, and conventional testing diagnoses. Because EUS and esophagogastroduodenoscopy (EGD) are uniformly performed in combination, the EUS diagnosis was rendered on the basis of previously established criteria combined with the EGD interpretation. The diagnoses then were compared to examine the contribution of EUS in this setting. RESULTS: The technique and defined criteria were easily applied to all subjects. All symptomatic patients had heartburn and were taking proton pump inhibitors (PPI). No asymptomatic patients were taking PPI. All diagnoses established with combined conventional testing were detected on EUS with upper endoscopy. Additionally, EUS resolved the etiology of a low lower esophageal sphincter pressure in two symptomatic patients and detected the additional diagnoses of slippage in two subjects. Among asymptomatic subjects, EUS identified additional diagnoses in two subjects considered to be normal by conventional testing methods. CONCLUSION: According to the findings, EUS is a feasible method for evaluating post-Nissen fundoplication hiatal anatomic relationships. The combination of EUS and EGD allows the mechanism of failure to be detected in patients presenting with postoperative symptoms after Nissen fundoplication.

Repeated sequences of interspike intervals in baroresponsive respiratory related neuronal assemblies of the cat brain stem.

Many neurons exhibit spontaneous activity in the absence of any specific experimental perturbation. Patterns of distributed synchrony embedded in such activity have been detected in the brain stem, suggesting that it represents more than "baseline" firing rates subject only to being regulated up or down. This work tested the hypothesis that nonrandom sequences of impulses recur in baroresponsive respiratory-related brain stem neurons that are elements of correlational neuronal assemblies. In 15 Dial-urethan anesthetized vagotomized adult cats, neuronal impulses were monitored with microelectrode arrays in the ventral respiratory group, nucleus tractus solitarius, and medullary raphe nuclei. Efferent phrenic nerve activity was recorded. Spike trains were analyzed with cycle-triggered histograms and tested for respiratory-modulated firing rates. Baroreceptors were stimulated by unilateral pressure changes in the carotid sinus or occlusion of the descending aorta; changes in firing rates were assessed with peristimulus time and cumulative sum histograms. Cross-correlation analysis was used to test for nonrandom temporal relationships between spike trains. Favored patterns of interspike interval sequences were detected in 31 of 58 single spike trains; 18 of the neurons with significant sequences also had short-time scale correlations with other simultaneously recorded cells. The number of distributed patterns exceeded that expected under the null hypothesis in 12 of 14 data sets composed of 4-11 simultaneously recorded spike trains. The data support the hypothesis that baroresponsive brain stem neurons operate in transiently configured coordinated assemblies and suggest that single neuron patterns may be fragments of distributed impulse sequences. The results further encourage the search for coding functions of spike patterns in the respiratory network.

Antigen-specific cancer immunotherapy using a GM-CSF secreting allogeneic tumor cell-based vaccine.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cell-based vaccines are currently one of the major forms of cancer vaccines. However, the preparation of GM-CSF-transduced autologous tumor vaccines is time-consuming and technically challenging. In addition, the host antigen presenting cells, rather than the tumor vaccine cells themselves, present tumor-specific antigens and prime the host T cells. Therefore, we tested the efficacy of antigen-specific allogeneic tumor vaccines. We used human papillomavirus 16 (HPV-16) E7 protein as a model tumor antigen, which is associated with the development of most cervical carcinoma. B16, a C57BL/6 (H-2(b)) derived melanoma cell line, was genetically engineered to produce GM-CSF alone (B16GM), HPV-16 E7 alone (B16E7), or both (B16GME7). These vaccine cells were injected into BALB/c (H-2(d)) mice (10(6) cells/mouse). Two weeks later, mice were challenged with 10(5) live HPV-16 E7(+) BL-1 (H-2(d)) tumor cells and monitored for tumor progression twice weekly. To determine the effective cell population in the antitumor immunity elicited by B16GME7, we carried out in vivo antibody depletion experiments using CD4 and CD8 specific antibodies. In addition, as a measure of the immune responses produced by B16GME7, we performed an in vitro cytotoxic T lymphocyte assay using a standard chromium release method. We found that all of the mice vaccinated with B16GME7 remained tumor free 49 days post-BL-1 challenge. In contrast, mice vaccinated with B16GM and B16E7 did not show any tumor protection against a similar dose of BL-1 cells. Furthermore, the antitumor immunity produced by B16GME7 was dependent on both CD4 and CD8 T cells. In addition, E7-specific cytotoxic T lymphocyte activity could be readily demonstrated in mice immunized with B16GME7. These results suggest that allogeneic tumor cells transduced with GM-CSF and the tumor antigen, HPV-16 E7, cannot only generate an E7-specific cytotoxic T lymphocytes response in vitro, but can also elicit a potent antitumor immune response against an E7 expressing tumor in vivo.

A noninvasive method for quantifying and distinguishing inflammatory skin reactions.

BACKGROUND: The ribonuclease protection assay (RPA) represents a technology that allows detection of small amounts of intact RNA. Recent progress in understanding cytokine networks in the skin suggests that measurements of cytokine mRNA levels could provide a method to distinguish various reactions such as irritant contact dermatitis and allergic contact dermatitis that can occur in the skin. OBJECTIVE: We attempted to differentiate and quantitate irritant and immunologic skin reactions by measuring mRNA levels. METHODS: We have used the technique of tape stripping human skin to remove superficial cell layers and have extracted RNA from these skin samples. This RNA was used for RPA analysis. RESULTS: By means of RPA analysis, we have demonstrated distinct cytokine profiles that appear to discriminate, for example, irritant from immunologic skin reactions. CONCLUSION: We have shown that multiple cytokine mRNA levels can be defined in these RNA samples obtained from the skin. This approach assesses not only the cytokine gene profiles, but at the same time may quantify the severity of common irritant versus allergic skin reactions.

Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16 E7 oncoprotein.

An important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported a recombinant vaccinia-based vaccine (Sig/E7/LAMP-1) that demonstrated significant anti-tumor effect in a subcutaneous tumor challenge model. In this study, we investigated the potency of the Sig/E7/ LAMP-1 vaccine in preventing and treating metastatic tumors. A tumor metastasis model was generated by injecting human papillomavirus type 16 (HPV-16) E6/E7 expressing tumor cells, designated TC-1, into the tail vein of syngeneic C57BL/6 mice. All the naive mice injected with 1 x 10(6) TC-1 cells developed tumors confined exclusively to the lungs within 1 month. For in vivo tumor prevention experiments, mice were vaccinated with Sig/E7/ LAMP-1 followed by tumor challenge. While tumor growth was observed in all of the mice (10/10) in the control groups, 8 of 10 vaccinated mice (80%) remained tumor-free 2 months post-tumor challenge. For in vivo treatment experiments, mice were first inoculated with TC-1 cells and then vaccinated with Sig/E7/ LAMP-1. Treatment with Sig/E7/LAMP-1 was effective in eliminating preexisting tumor cells in 4 of 5 vaccinated mice. Most importantly, treatment with Sig/E7/LAMP-1 resulted in regression of fully established lung tumors in 10% (5/10) of vaccinated mice. Our data suggest that the Sig/E7/LAMP-1 vaccine is effective in controlling the hematogenous spread of TC-1 tumor cells. In addition, the TC-1 lung metastasis model can be used to test the efficacy of various E6/E7-specific vaccines and immunotherapeutic strategies.

Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3.

The rat basophilic leukemic (RBL-2H3) cell line was stably transfected with the endogenously expressed Ca2+-dependent protein kinase C-alpha (PKC-alpha) and -betaI and the Ca2+-independent delta and epsilon isoforms to study their functional roles. In addition, the Ca2+-independent PKC-eta was expressed. All transfected PKC isoforms translocated to the membrane-containing fraction in response to aggregation of the IgE-sensitized high affinity receptor for IgE (Fc epsilonRI) with the Ag dinitrophenyl(25)-BSA. All PKC transfectants, except PKC-eta, showed increased proliferative responses, and aggregation of Fc epsilonRI further enhanced the rate of proliferation. The PKC transfectants also showed increased phosphoinositide hydrolysis in response to Ag aggregation of receptors. No marked differences in the Ca2+ responses of the transfectants to Ag or thapsigargin were observed. Overexpression of PKC-alpha or -epsilon specifically inhibited receptor-dependent cytosolic phospholipase A2 (cPLA2) activity, whereas this activity was enhanced in the PKC-betaI transfectant. Analysis of the secretory response revealed that overexpression of PKC-betaI and -eta significantly enhanced secretion. A broad spectrum of cytokine mRNAs was detected in all transfectants, and overexpression of PKC-betaI significantly enhanced the receptor-dependent production of IL-2 and IL-6 mRNA. These studies identify PKC-alpha and -epsilon as negative regulators of cPLA2 activity and demonstrate the importance of PKC-beta as a positive modulator of secretion, cPLA2 activity, and cytokine production in this mast cell line.