B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

T-cell variant of classical Hodgkin's lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction

The atypical cells of CD30(+) cutaneous lymphoproliferative disorders (CD30CLD) are commonly of T-cell origin and frequently have a similar morphology as Hodgkin or Reed-Sternberg cells of Hodgkin's lymphoma (HL). HL is one of the tumors associated with CD30CLD. Although most studies support a B-cell derivation of the tumor cells in HL, recently a few cases of classical HL with T-cell genotype have been reported. We report a patient who presented with CD30CLD whose lymph nodes showed classical HL of mixed cellularity subtype at presentation. By single-cell PCR, the same clonal gene rearrangements of the T cell receptor-beta gene locus could be assigned to the CD30(+) and CD15(+) cells of both skin and lymph node. In a lymph node biopsy specimen taken in relapse after several courses of chemotherapy, the CD30(+) tumor cells were abundant. The T cell-derived tumor cells displayed aberrant expression of the Pax-5 gene in all specimens. A common clonal origin of both CD30CLD and HL of the lymph node in the patient presented here suggests that HL with T-cell genotype exists in association with CD30CLD as well as in sporadic cases and may share clonal origin with the skin tumor.     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

Significance of fibrils in the formation of the Kimmelstiel-Wilson nodule

Summary The pathogenesis of the nodular lesion in diabetic glomerulosclerosis is described in association with fibrils. Thirteen diabetic patients with glomerular nodular lesions and 9 diabetics without the nodules were examined by electron microscopy using periodic acid-thio-carbohydrazide-silver proteinate staining. In cases of nodular glomerulosclerosis, abundant fibrillar structures mixed with electron-dense material were detected within the nodule and the mesangial matrix. They were also occasionally observed along the subendothelial space of the glomerular capillary walls. On the cross-section, these fibrils, including the lucent periphery, were 34 nm wide. Immunohistologically, collagen V and collagen VI were detected in nodular lesions. In contrast, in cases of the diffuse type of glomerulosclerosis, the widened mesangium was composed of dense material, which resembled the original mesangial matrix. The above fibrils were not detected in the mesangium. These findings suggest that the accumulation of the peculiar fibrils in the glomerular mesangium is a major pathogenic factor in the formation of Kimmelstiel-Wilson nodules.     

Lamivudine and IFN-beta sequential therapy in HBe antigen-positive patients with chronic hepatitis B virus genotype C infection.

Sequential treatment with lamivudine and interferon (IFN) has induced sustained biochemical and virologic responses in the majority of patients with chronic hepatitis B in France. However, the efficacy of sequential treatment in patients with chronic hepatitis B virus (HBV) genotype C infection has not been evaluated. Twenty-four HBe antigen-positive patients were treated with 100 mg lamivudine alone for 16-32 weeks, then with both 6 MU IFN-beta and lamivudine for 4 weeks, and lastly with IFN-beta alone for 20 weeks. Sustained response was achieved in 7 (29%) patients 24 weeks after the end of therapy. No lamivudine-resistant variants emerged in any patient. Hepatitis flare occurred in 3 patients after the withdrawal of lamivudine, but none had decompensation. The patients with sustained response were significantly younger at baseline (p = 0.033) and had a significantly lower HBV DNA level at the start of IFN (p = 0.020) than those without sustained response. In conclusion, the rate of response to sequential therapy with lamivudine and IFN in HBe antigen-positive patients with HBV genotype C infection was lower than the rate reported previously. Patients who were young or who had a favorable virologic response to lamivudine were more likely to have a sustained response.     

Mechanism of bonelike apatite formation on bioactive tantalum metal in a simulated body fluid.

Development of tantalum metal with bone-bonding ability is paid much attention because of its attractive features such as high fracture toughness, high workability and its achievement on clinical usage. Formation of bonelike apatite is an essential prerequisite for artificial materials to make direct bond to living bone. The apatite formation can be assessed in vitro using a simulated body fluid (SBF) that has almost equal compositions of inorganic ions to human blood plasma. The present authors previously showed that the apatite formation on tantalum metal in SBF was remarkably accelerated by treatment with NaOH aqueous solution and subsequent firing at 300 degrees C, while untreated tantalum metal spontaneously forms the apatite after a long soaking period. The purpose of the present study is to clarify the reason why the NaOH and heat treatments accelerate the apatite formation on tantalum metal. X-ray photoelectron spectroscopy was used to analyze changes in surface structure of the tantalum metal at an initial stage after immersion in SBF. Untreated tantalum metal had tantalum oxide passive layer on its surface, while amorphous sodium tantalate was formed on the surface of the tantalum metal by the NaOH and heat treatments. After soaking in SBF, the untreated tantalum metal sluggishly formed small amount of Ta-OH groups by a hydration of the tantalum oxide passive layer on its surface. In contrast, the treated tantalum metal rapidly formed Ta-OH groups by exchange of Na+ ion in the amorphous sodium tantalate on its surface with H3O+ ion in SBF. Both the formed Ta-OH groups combined with Ca2+ ion to form a kind of calcium tantalate, and then with phosphate ion, followed by combination with large amount of Ca2+ ions and phosphate ions to build up apatite layer. The formation rate of Ta-OH groups on the treated tantalum metal predominates the following process including adsorption of Ca2+ ion and phosphate ion on the surface. It is concluded that the acceleration of the apatite nucleation on the tantalum metal in SBF by the NaOH and heat treatments was attributed to the fast formation of Ta-OH group, followed by combination of the Ta-OH groups with Ca2+ and phosphate ions.     

Gallbladder adenocarcinoma arising in Rokitansky–Aschoff sinus

Carcinoma arising from Rokitansky–Aschoff sinus (RAS) is extremely rare; only eight cases have been reported in the literature. Herein is reported a case of minute adenocarcinoma arising in RAS. A 77‐year‐old Japanese man with gallbladder stones underwent cholecystectomy. A tiny submucosal tumor (1 cm × 1 cm) was incidentally recognized. Histologically, the submucosal tumor was located in the subserosa and, to a lesser extent, in the fibromuscular layer. It was adenocarcinoma. RAS were recognized within the tumor, and there was a gradual transition between RAS and the adenocarcinoma. Mucin histochemistry indicated neutral and acidic mucins in the cytoplasm and lumens of the adenocarcinoma cells. Immunohistochemistry showed that the adenocarcinoma cells were positive for cytokeratin, epithelial membrane antigen, carbohydrate antigen 19‐9, K‐i67 (labeling = 80%), MUC1, MUC5AC and MUC6. In contrast, the adenocarcinoma cells were negative for CEA, c‐erbB2, p53 protein, MUC2 and CD10. In summary, minute subserosal adenocarcinoma, which arose in RAS, was found incidentally; therefore careful examination of resected gallbladders is necessary.     

Low-grade fibromyxoid sarcoma arising in the big toe

Low-grade fibromyxoid sarcoma (LGFMS) is a rare tumor. Reported herein is a case of LGFMS arising in the big toe. The patient was a 58-year-old man who underwent excision of the tumor. The tumor was well-demarcated. Histologically, there were proliferating spindle-shaped tumor cells arranged in a whorled growth pattern, and the stroma showed hyalinized collagen bundles and a myxoid matrix. Nuclear mitotic figures were conspicuous in part. A large rosette-like structure with hyalinized stroma was found, which is characteristic of LGFMS. The differential diagnosis included tumor occurrence in adults; tending to arise in distal extremities; and having bland fibromyxoid histological features, such as fibroma of tendon sheath, low-grade myxofibrosarcoma and acral myxoinflammatory fibroblastic sarcoma. It was not possible to detect the FUS/CREB3L2 and FUS/CREB3L1 fusion genes from the formalin-fixed and paraffin-embedded tissue, although the histological features of the present case were typical of LGFMS. LGFMS may become more common with time, and unique cases may accumulate.     

Expression and intracellular localization of FKHRL1 in mammary gland neoplasms

FKHRL1 (FOXO3a), a member of the Forkhead family of genes, has been considered to be involved in the development of breast tumors; however, the in vivo expression and activation status of FKHRL1 in breast tumors still remains unclear. We immunohistochemically demonstrated the expression and intracellular localization of FKHRL1 in human breast tumors by the novel anti-FKHRL1 antibody which is available for formalin-fixed paraffin-embedded specimens. In a total of 51 cases of benign tumors, FKHRL1 was diffusely expressed in all cases, and its intracellular localization was revealed to be cytoplasmic (inactive form) in 94% of cases of intraductal papillomas (16/17) and 91% cases of fibroadenomas (31/34), with a similar pattern to normal glandular epithelium. In invasive ductal carcinomas, 83% of the cases (93/112) diffusely expressed FKHRL1; however, unlike benign tumors, 71% of the cases (66/93) showed the nuclear-targeted, active form of FKHRL1. Moreover, activated FKHRL1 was predominantly observed in scirrhous (29/36, 81% of the cases) and papillotubular (30/38, 79% of the cases) subtypes, compared to the solid-tubular subtype (7/19, 37% of the cases). Furthermore, the cases with nuclear-targeted FKHRL1 showed a tendency to have lymph nodal metastasis with statistical significance (P < 0.0001). Thus, the activation of FKHRL1 seems to be recognized as one of the specific features of invasive ductal carcinoma of the breast.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.