Structural and functional analysis of the two human tumor necrosis factor receptors

Pleiotropic TNF activities are mediated through the two cell surface receptors (TNFRs). Both are transmembrane proteins sharing 30% protein sequence homology in their extracellular domains, but little homology in intracellular domains. It suggests that the two receptors interact with separate signal molecules which mediate distinct biological activities. The extracellular domain of TNFRs are characterized by 4 conserved cysteine-rich motif repeat domains. In order to identify the ligand-binding domain(s) of the two TNFRs, recombinant domain-deletion mutants of TNFRs were expressed and their binding capacities assessed by ligand blot analysis and Scatchard analysis. Our results are consistent with those from x-ray cinematography, that domain 3 of both TNFRs is critical for TNF-$\alpha$ binding. But the domain 4 of TNFR-2, but not that of TNFR-1, is also important for binding. Whether the domain 4 of TNFR-2 is involved directly in binding TNF, or indirectly in the correct folding of other domains of the receptor, is still unknown. To investigate the functional species specificity of TNFR-1, we transfected an inducible hTNFR-1 expression construct into murine L929 cells. These established L929 cells, are about as sensitive to hTNF-$\alpha$ as parental cells, in the non-induced state. However, they become 25-100 fold more sensitive to hTNF-$\alpha$ after hTNFR-1 expression is induced. \sp{125}I-TNF-$\alpha$ saturation binding assay and Scatchard analysis showed that the transfected hTNFR-1, interacts with hTNF-$\alpha$ much more strongly than mTNFR-1 does. Taken together, these results demonstrate homologous species preference of TNFR-1. The advantage of inducible hTNFR-1 expression and the significance of these cell lines on clinical research for sensitive and reproducible hTNF bioassay are discussed. The functional roles of the two TNFRs in mediating NF-$\kappa$B activation, and inducing IL-2R$\alpha$ chain (CD25) and ICAM-1 (CD54) expression, were examined using receptor-specific antisera and TNF mutants, Northern blot analysis, gel shift assay and flow cytometry on human NK-like YT cells. The results showed that antisera against the two hTNFRs and human TNF mutants binding exclusively or preferentially to one of the two TNFRs are all capable of triggering NF-$\kappa$B activation and CD25/CD54 nduction. In combination, the two receptor antisera produce synergistic responses. Northern blot analyses indicated that the mRNAs of p50 and p52, but not that of p65 RelA, are induced by TNF or either of the two TNFR antisera. Supershift assay identified that the nuclear NF-$\kappa$B complexes activated by TNF are P50/p50 and p50/p65 dimers. Finally, a TNF triggered homoaggregation on YT cells was observed. We showed that TNFR-2 mediates this phenomenon much stronger than TNFR-1 does. Taken together, these results indicated that NF-$\kappa$8 activation, homoaggregation, and the induction of CD25 and CD54 on YT cells could be mediated independently through individual TNFRs via separate signaling pathways. Finally, by successful identification of TNF-induced genes, including MCP-1 (monocyte chemoattractant protein-1), in human monocyrtic leukemia cells ML-1a using a modified differential display technique, we showed that this new technique is a powerful tool in identifying TNF-regulated genes involved in different biological activities

Primary Hyperparathyroidism With Brown Tumor Mimicking Metastatic Bone Malignancy

Bone and joint pain are commonly encountered conditions in daily practice. In the elderly, when osteolytic lesions are identified in imaging studies, metastatic bone tumor is the first impression that comes to the clinician's mind. Although the worst-case scenario should be ruled in, other differential diagnoses such as metabolic bone disease should be considered as well. We report a case of brown tumor caused by parathyroid adenoma. The patient had initial presentation of diffuse bone pain and multiple osteolytic lesions on imaging studies similar to metastatic bone tumor. With a systematic approach and awareness of metabolic bone disease, an accurate diagnosis was finally reached. Appropriate treatments, including preventive internal fixation of the impending femoral fracture and surgical excision of the parathyroid adenoma were performed accordingly. The key treatment for the condition was surgical excision of the parathyroid adenoma. After normalization of serum intact-parathyroid hormone level, the bony lesions resolved and required no further orthopedic surgery. The patient is now symptom-free. In addition to suspecting malignancy, the clinician should be highly alert to other possible causes of bony lesions. Brown tumor should be kept in mind during daily practice

Clinical outcomes and prognostic factors of Ewing sarcoma: A clinical analysis of 12 patients in Taiwan

Background: Ewing sarcoma is extremely rare in people from East and Southeast Asia. Methods: The records of 12 patients diagnosed with primary Ewing sarcoma and treated at our institution from 1997 to 2009 were retrospectively reviewed. Results: There were seven male and five female patients and their mean age at diagnosis was 22 years (range, 12–48 years). Two patients (16.7%) had distant metastasis at diagnosis. The primary tumor sites were the trunk in seven patients (58.3%) and the extremities in five patients (41.7%). Eleven patients received neoadjuvant chemotherapy followed by wide excision surgery, and then adjuvant chemotherapy. One patient received only chemotherapy without surgical intervention due to poor cardiac and pulmonary function. At a mean follow-up of 33 months, the 2-year overall survival rate (OS) was 45.5%. Distant metastasis was the only statistically significant prognostic factor of OS in our study. The 2-year OS rates of patients with lung metastasis and without lung metastasis were 0% and 42.9%, respectively (p = 0.021). The t(11;22)(q24:q12) translocation was present in all patients in our series. Conclusion: We confirmed that distant metastases is highly predictive of a poor outcome, and that the t(11;22)(q24:q12) translocation was present in all patients in our series