The possible involvement of immunoglobulin G4 (IgG4) in the pathogenesis of idiopathic sclerosing lesions has been suggested. In this study, a clinicopathologic analysis was performed to reveal characteristics of retroperitoneal fibrosis relating to IgG4. The study involved 17 patients with retroperitoneal fibrosis. Immunohistochemistry revealed numerous IgG4-positive plasma cell infiltrates in 10 cases (IgG4-related), but only a few positive cells in 7 cases (non-IgG4-related). All patients with IgG4-related retroperitoneal fibrosis were male, whereas all except 1 with unrelated lesions were female. Histologically, eosinophilic infiltration (>5 cells per high-power field) and obliterative phlebitis were commonly observed in IgG4-related lesions. Serologically, serum IgG and IgG4 concentrations were significantly higher in the IgG4-related cases, with the IgG4 concentrations all over 135 mg/dL (the upper limit of the normal range). Steroid therapy was performed in 13 cases, and was effective irrespective of IgG4. Three patients had recurrence during the follow up. Five of 10 IgG4-related cases had sclerosing lesions at other sites. The only tests that reliably distinguish the 2 groups were serum IgG4 levels or IgG4/IgG ratio in the plasma cells in a tissue biopsy. The only major clinical difference was the striking male predominance in IgG4-related cases. In conclusion, this study revealed that retroperitoneal fibrosis could be classified as IgG4-related or not. This distinction seems important to help better characterize the biology/pathogenesis of both groups and better predict the possibility of other IgG4-related processes at other anatomic sites.

Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H&E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.

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Distinguishing a well-differentiated hepatocellular carcinoma (HCC) from normal and cirrhotic liver tissue or benign liver nodules, such as hepatic adenoma (HA) and focal nodular hyperplasia (FNH), may be very difficult in some cases, particularly in small needle core biopsies. We studied the expression of Glypican-3 (GPC3) and CD34 in 107 cases of HCC, 19 cases of HA, and 16 cases of focal nodular hyperplasia (FNH). In addition, we studied GPC3 expression in 225 cases of nonhepatic human tumors with epithelial differentiation. Ninety-four of 107 cases (88%) of HCC showed focal or diffuse cytoplasmic GPC3 staining, whereas all HA and FNH cases were GPC3-negative, and only 7 of 225 cases (3%) of nonhepatic tumors with epithelial differentiation expressed GPC3. The sensitivity and specificity of GPC3 for HCC was 88% and 97%, respectively. There were three CD34 staining patterns observed in hepatic tissue: negative, incomplete positive, and complete positive. In negative staining pattern, only blood vessels in portal triads or rare sinusoidal spaces immediately adjacent to portal tracts were positive. The negative staining pattern was seen in normal or cirrhotic liver tissue only. The complete CD34 staining pattern showed virtually all sinusoidal spaces with CD34-positive staining throughout the lesion. The complete CD34 staining pattern was seen in virtually all cases of HCC and in only some cases of HA and FNH. The incomplete CD34 staining pattern was characterized by either CD34 positivity in virtually all sinusoidal spaces in some but not all nodules or CD34 positivity in the peripheral sinusoidal spaces adjacent to portal triads. The incomplete CD34 staining pattern was seen in rare cases of HCC and in most cases of HA and FNH. We conclude that GPC3 is a very specific marker not only for differentiating HCC from nonhepatic tumors with epithelial differentiation, but also for differentiating HCC from HA and FNH. GPC3 immunoreactivity, in combination with a complete CD34 immunostaining pattern, greatly facilitates the accuracy of distinguishing between malignant hepatic lesions and benign mimickers.

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet-derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)␤ gene by Cre-loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF-A, -B, -C, and -D as well as PDGFR␣ and ␤ was detected. Depletion of PDGFR␤ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT-PCR. PDGF-BB, but not PDGF-AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF-BB were eliminated by depletion of PDGFR␤ in MSCs except that PDGF-BB still suppressed osterix expression in PDGFR␤-depleted MSCs. Depletion of PDGFR␤ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFR␤ gene deletion, we showed that PDGFR␤ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFR␣ on the osteogenic differentiation were very subtle. PDGFR␤ could represent an important target for guided tissue regeneration or tissue engineering of bone.