Observation and extirpation of a giant-size type-B2 thymoma IIb with its histological, macroscopic, and computer tomogram correlate, and literature review.

This report describes the interdisciplinary approach and solving algorithm of a DIN 9001:2000 certified tumor board in managing a giant-size type-B2 thymoma IIb in an elderly patient. The process of managing the thymoma with specialists of surgery, internal medicine, radiology, and pathology until finally extirpation and continuous follow-up is described. Respective computerized tomography scans, histology, macro-pathology, and operative pictures of the case are provided as well as an upto-date literature review.

Expression of proteasomal immunosubunit beta1i is dysregulated in inflammatory infiltrates of minor salivary glands in Sjogren's syndrome.

OBJECTIVE: Minor salivary gland specimens were analyzed to investigate dysregulation of the proteasome system in patients with Sjögren's syndrome (SS) and patients with sicca syndrome. METHODS: Labial biopsy specimens from 17 patients with SS and 11 patients with non-autoimmunesicca syndrome were analyzed by immunohistochemistry for expression of the inducible proteasomal subunits ss1i, ss2i, and ss5i. The infiltrating subsets of lymphocytes were characterized using immunofluorescence stainings against the cell-surface markers CD20 and CD27. Two-dimensional electrophoresis and immunoblotting were used for detection of the proteasomal subunits ss1 and ss1i in peripheral blood monocyte cells. Gene expression of the constitutive subunits ss1, ss2, and ss5 and the corresponding inducible subunits ss1i, ss2i, and ss5i was further investigated at the mRNA level in small lip biopsies using real-time polymerase chain reaction. RESULTS: The expression of ss1i in infiltrating and peripheral immune cells was altered in patients with SS compared to patients with non-autoimmune sicca syndrome and healthy controls. No significant differences were found in ss2i and ss5i expression between the same groups in small lip biopsies. Chisholm-Mason grade and ss1i expression were found to be inversely correlated (Spearman r = -0.461, p = 0.014). The phenotype and distribution of the lymphocytic infiltrate showed no differences between patients with primary and secondary SS regardless of ss1i expression. CONCLUSION: The proteasomal ss1i subunit is dysregulated in peripheral white blood cells and in inflammatory infiltrates of minor salivary glands in patients with SS.

Cleft lip and/or palate with monogenic autosomal recessive transmission in Pyrenees shepherd dogs.

OBJECTIVE: To document the genetic background of Pyrenees shepherd dogs as it relates to the incidence of cleft lip and/or cleft palate, to describe the phenotype, and to determine possible candidate genes. DESIGN: Pedigree analysis was performed and blood samples were taken from five affected pups, their siblings, and parents. Seven candidate genes were selected and linkage analysis was performed. Further methods used included sequencing and histology. RESULTS: In 37 litters consisting of 163 pups, we found 47 affected pups in a total population of 2104. The male:female ratio was 1:0.96. Affected pups showed isolated cleft lip and/or cleft palate; no attendant disorders have been reported. Despite a high degree of relationship, two affected pups displayed a cleft palate (- H S H -) and a cleft lip with or without cleft palate (L A -) cleft formation. Histology of affected pups showed that the medial edge epithelium remained intact and did not undergo an epithelial-mesenchymal transformation. There was no evidence for linkage between the trait and TGFb3 or Msx1. Subsequent sequencing excluded the coding sequence of Fst as well. CONCLUSION: Pedigree analysis showed that cleft palate is not genetically distinct from cleft lip with or without cleft palate but is inherited in this breed as a monogenic autosomal recessive trait. Linkage analysis and sequencing excluded TGFb3, Msx1, and Fst as candidate genes. Histology of affected pups showed that the medial edge epithelium is still intact.

CD146 protein in prostate cancer: revisited with two different antibodies.

AIMS: CD146 is a potentially metastasis promoting cell adhesion molecule and its expression has been described in various solid tumours. We aimed to evaluate the expression of CD146 in prostate cancer by immunohistochemistry in a clinically characterised study cohort to evaluate its prognostic properties. METHODS: We evaluated the CD146 protein expression using a polyclonal and a monoclonal antibody on 169 clinico-pathologically characterised cases. Statistical analyses were applied to test for correlations and diagnostic and prognostic associations. RESULTS: CD146 detection with the polyclonal antibody revealed marked differential expression between tumour and normal tissue and was also a significant marker for shortened PSA relapse free survival. The monoclonal CD146 antibody demonstrated a weaker epithelial signal, which was significantly correlated with that of the polyclonal antibody, but revealed no prognostic value. However, the Western blot of the polyclonal antibody displayed a clearly reduced specificity. CONCLUSIONS: Evaluation of protein expression can be highly dependent on the primary antibody employed. A credible evaluation of antibody specificity is crucial to prove the validity of protein expression studies. The immunoreactivity of the polyclonal CD146 antibody (Abcam, ab28360) is prognostic of PSA-relapse in prostate cancer patients, although its immunoreactivity is possibly not restricted to CD146 associated epitopes.

Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice.

Galectins have recently emerged as central regulators of the immune system. We have previously demonstrated that carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Here, we investigate the potential therapeutic effect of galectin-2 in experimental colitis. Galectin-2 expression and its binding profile were determined by immunohistochemistry. Acute and chronic colitis was induced by dextran sodium sulfate administration and in a T-cell transfer colitis model. Apoptosis was assessed by TdT-mediated dUTP-biotin nick-end labeling, and cytokine secretion was determined by cytometric bead array. We show that galectin-2 was constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated. Our study provides evidence that galectin-2 induces apoptosis in vivo and ameliorates acute and chronic murine colitis. Furthermore, galectin-2 has no significant toxicity over a broad dose range, suggesting that it may serve as a new therapeutic agent in the treatment of inflammatory bowel disease.

KPNA2 protein expression in invasive breast carcinoma and matched peritumoral ductal carcinoma in situ.

The aim of this study was to evaluate protein expression of Karyopherin alpha 2 (KPNA2) in invasive breast cancer and matched ductal carcinoma in situ (DCIS) and to correlate it with clinicopathological data, including patient survival. KPNA2 protein expression was assessed by immunohistochemistry in breast tissue samples, containing invasive carcinomas, DCIS, and adjacent histologically benign breast tissues. A polyclonal goat KPNA2 antibody was used for immunostaining of 83 clinicopathologically characterized cases. For statistical analysis, staining of at least 10% of nuclei was considered KPNA2 positive. Immunohistochemical detection of KPNA2 in invasive carcinoma showed a significant correlation with higher tumor stage, positive lymph node status, higher tumor grade, and negative ER status. Concordantly, KPNA2-positive tumors (31.3%) showed significantly shorter disease-free survival times (69 months vs 118 months; p = 0.007). KPNA2 protein expression was also detected in DCIS (21.3%) adjacent to invasive tumor and correlated with nuclear grade (p = 0.013). Expression of KPNA2 in invasive breast cancer correlates with conventional prognostic parameters and shorter disease-free survival. Additionally, KPNA2 is overexpressed in DCIS, particularly high grade lesions, which emphasizes its potential role in carcinogenesis of invasive breast carcinomas.

Tissue pretreatment with formic acid might lower HercepTest scores in breast cancer.

Creutzfeldt-Jakob disease and other prion diseases are diseases with yet not well-defined routes of transmission and infection. The safe processing of potentially contaminated tissue material remains a challenge for histologic laboratories. Formic acid pretreatment is considered to be effective in prion inactivation. We evaluated the c-erbB2 and the hormone receptor-status in potentially prion infectious breast cancer tissue after pretreatment with formic acid. Paired breast cancer tissue samples were immunostained with commercially available antibodies against c-erbB2, estrogen receptor, and progesterone receptor with 1 tissue sample of each pair being pretreated with 98% formic acid. Staining was evaluated either according to the HercepTest score or using an immunoreactive score. Additionally, fluorescence in situ hybridization (FISH) analyses were performed for 7 of these cases. Untreated tissues showed strong circumferential staining for c-erbB2 (HercepTest score 3+), whereas the membranous staining of the tissues pretreated with formic acid was significantly weaker. FISH analyses showed no differences in both groups. The hormone receptor expression was not significantly influenced and positivity was maintained in all cases. In breast cancer patients, the pretreatment of tissue with formic acid for prion-decontamination in the case of suspected Creutzfeldt-Jakob disease or other prion diseases can lead to underestimation of the immunohistologically determined c-erbB2 status. In these cases, a c-erbB2-FISH analysis should be performed. For the immunostaining of hormone receptors in breast cancer, formic acid pretreatment can be applied without negative effects on the sensitivity or specificity of the assay.

Anaplastic large-cell non-Hodgkin's lymphoma of the breast in periprosthetic localisation 32 years after treatment for primary breast cancer--a case report.

Primary, as well as secondary, lymphomas of the breast are rare diseases and might, in some cases, be misdiagnosed as breast cancer on routine hematoxylin/eosin stainings. We report a case of an anaplastic large cell lymphoma in a 72-year-old woman with a history of breast cancer treated with breast-ablative surgery and a subsequent silicon implant 32 years ago. Clinically, she presented with an ulceration of the skin, which had developed within a few months. On conventional histology, the tumor cells were mimicking poorly differentiated invasive ductal carcinoma with a prominent leukocytic infiltrate. The immunoprofile of the tumor showed negativity for cytokeratins and led to the diagnosis of a CD30-positive anaplastic large cell lymphoma.

C4d deposits mark sites of meniscal tissue disintegration.

Although the frequent occurrence of meniscal degeneration is a well-known fact and its consequences include rupture and even loss of the meniscus, the pathogenetic factors are established insufficiently. Because complement factors and leukocytes are present in synovial fluid, we tried to detect complement deposits and macrophages in menisci, which displayed degenerative changes. We therefore performed a retrospective analysis by immunohistochemical staining of C4d and CD68 in meniscal tissue derived from patients (n=15) who underwent meniscectomy because of meniscal tears and from three autopsy cases (n=3). In this study, focal C4d deposits in the meniscal extracellular matrix in areas of mucoid degeneration or fibrillation were demonstrated for the first time, while no C4d deposits in the avascular zone of menisci without signs of degeneration or injury could be detected. In addition, colocalization of C4d and CD68+ cells was found at sites of meniscal tissue disintegration in five cases. These results represent the first evidence for an involvement of complement and macrophages in meniscal tissue disintegration, indicating a complement mediated reaction at the site of tissue alteration.

24-h storage of pig livers in UW, HTK, hydroxyethyl starch, and saline solution: is microdialysis an appropriate method for the continuous graft monitoring during preservation?

Recent studies demonstrate the feasibility of microdialysis to monitor metabolism in ischemic livers. Whether these parameters correlate with markers of liver cell integrity in an experimental model using pig livers and different preservation solutions was an aim of this study. Pig livers were flushed with either 4 degrees C Histidine-Typtophan-Ketoglutarate solution (HTK) (Custodiol), University of Wisconsin solution (ViaSpan), and hydroxyethyl starch, or 12 degrees C saline solution. After 24-h storage, the livers were rinsed with saline to measure liver enzymes and lactate from the effluate. Utilizing microdialysis, intraparenchymal lactate, pyruvate, glucose, and glycerol was monitored. Tissue biopsies were taken for histological examinations. Cold preservation resulted in a decrease of metabolic activity measured by intrahepatic glucose, lactate, and pyruvate levels, as well as lactate in the effluate, independently of the solution used. Of particular interest, glycerol levels partially reflected the extent of hepatocellular damage and liver enzyme release. Glycerol levels partially discriminated preservation of different quality and were in accordance to histological findings and liver enzyme release. Lactate, pyruvate, and glucose levels were not appropriate as markers during cold storage. Whether or not glycerol monitoring could represent an additional and rational complementation to the current practice of macroscopic, microscopic and donor evaluation has to be clarified by further studies.

Tissue-specific up-regulation of the proteasome subunit beta5i (LMP7) in Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by autoimmune infiltration and focal accumulation of lymphocytes in the exocrine glands, with a predominance of CD4-positive T cells. Since these histologic findings are nonspecific, determination of clinical and serologic abnormalities contribute to the diagnosis. The aim of this study was to identify a novel, disease-specific, immunologically relevant marker for SS. METHODS: To analyze disease-related and tissue-specific expression of candidate markers, we examined biopsied minor salivary glands and peripheral blood mononuclear cells from patients with primary and secondary SS (n = 26) as well as from patients with sicca symptoms without autoimmune sialadenitis (n = 15). Expression of the Th1/Th2-related chemokines CCL3 (macrophage inflammatory protein 1alpha) and CCL2 (monocyte chemoattractant protein 1), CXCL7 (neutrophil-activating peptide 2 [NAP-2]), interleukin-1beta, inducible costimulator, and the proteasome subunits alpha3 (C9) and beta5i (LMP7) was analyzed at the messenger RNA (mRNA) level using real-time polymerase chain reaction techniques. Immunohistochemical analysis was used to identify the beta5i (LMP7)-expressing cell populations in minor salivary glands. RESULTS: The expression profiles revealed a significant up-regulation of beta5i (LMP7) exclusively in the salivary glands of SS patients. Immunohistochemistry confirmed expression of the immunoproteasome subunit beta5i (LMP7) within the acinar and ductal epithelial cells. No significant difference in the distinct histologic focus scores was evident for the expression of the markers investigated. In the peripheral blood compartment, the expression of CXCL7 was up-regulated both in primary and in secondary SS. CONCLUSION: Tissue-specific up-regulation of beta5i (LMP7) mRNA was shown to be characteristic of SS, indicating a disease-specific modulation of the proteasome system. Expression of beta5i (LMP7) represents an independent parameter that can be used in addition to the focus score to distinguish SS in biopsied labial salivary glands.

Prognostic relevance of AGR2 expression in breast cancer.

PURPOSE: We aimed to evaluate the expression of the human anterior gradient-2 (AGR2) in breast cancer on RNA and protein level and to correlate it with clinicopathologic data, including patient survival. EXPERIMENTAL DESIGN: AGR2 mRNA expression was assessed by reverse transcription-PCR in 25 breast cancer samples and normal tissues. A polyclonal rabbit AGR antiserum was used for immunohistochemistry on 155 clinicopathologically characterized cases. Statistical analyses were applied to test for prognostic and diagnostic associations. RESULTS: Immunohistochemical detection of AGR2 was statistically significantly associated with positive estrogen receptor status and lower tumor grade. AGR2-positive tumors showed significantly longer overall survival times in univariate analyses. For the subgroup of nodal-negative tumors, an independent prognostic value of AGR2 was found. CONCLUSIONS: The expression of AGR2 in breast cancer is strongly associated with markers of tumor differentiation (estrogen receptor positivity, lower tumor grade). A prognostic effect of AGR2 for overall survival could be shown, which became independently significant for the group of nodal-negative tumors.

Giant cell tumors of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR.

Giant cell tumors (GCTs) of the bone are osteolytic neoplasms with variable degrees of aggressiveness. The aim of this study was the molecular characterization of GCT tissue. We established gene expression profiles and discovered a number of genes that have not been described in GCTs before. RNA was prepared from 7 cryopreserved GCTs (primary tumors n = 5, relapses n = 2) and was hybridized to Affymetrix HG U133A microarrays. Paraffin-embedded samples were used for immunohistochemical validation (primary tumors n = 16, relapses n = 6). Gene ontology revealed that the majority of genes, found to be differentially expressed between primary and recurrent GCTs, were associated with receptor tyrosine kinase activity. We selected one upregulated gene (Claudin 7) and four downregulated genes (CD52, Ephrin A1 receptor, autocrine motility factor receptor [AMFR] and fibroblast growth factor receptor 3 [FGFR3] for further analysis using immunohistochemistry. Immunohistochemical analysis of CD52, AMFR, and Ephrin A1 receptor revealed expression profiles concordant with the microarray data, also with regard to differences between primary tumors and relapses.

Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo.

Regulatory T cells (Tregs) play a fundamental role in the suppression of different immune responses; however, compartments at which they exert suppressive functions in vivo are unknown. Although many groups have described the presence of Tregs within inflammatory sites, it has not been shown that inflamed tissues are, indeed, the sites of active suppression of ongoing immune reactions. Here, by using alpha(E)+ effector/memory-like Tregs from fucosyltransferase VII-deficient animals, which lack E/P-selectin ligands and fail to migrate into inflamed sites, we analyzed the functional importance of appropriate Treg localization for in vivo suppressive capacity in an inflammation model. Lack of suppression by Tregs deficient in E/P-selectin ligands demonstrates that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues. In contrast, control of proliferation of naive CD4+ T cells during the induction phase of the immune response is more efficiently exerted by the naive-like alpha(E)-CD25+ Treg subset preferentially recirculating through lymph nodes when compared with its inflammation-seeking counterpart. Together, these findings provide the first conclusive evidence that appropriate localization is crucial for in vivo activity of Tregs and might have significant implications for anti-inflammatory therapies targeting recruitment mechanisms.

Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection.

Almost no data exist concerning the role of antibody-mediated mechanisms in human acute cellular liver allograft rejection (ACR). Therefore, the aim of this study was to determine whether ACR is associated with depositions of complement split products and increased infiltration by B-lymphocytes, plasma cells and macrophages. A total of 35 liver biopsy specimens (ACR n=22, controls n=13) were analyzed by immunohistochemical single and double staining. The average numbers of CD 20(+), CD 38(+) and CD 68(+) cells per portal tract were established while the presence of C4d and C3d deposits was evaluated semiquantitatively. Significantly greater numbers of CD 20(+) (P=0.029) and CD 38(+) (P=0.014) cells were found in the ACR specimens than in the control specimens. Additionally, 50% of patients diagnosed with ACR showed C4d deposits along portal capillaries, which was associated with a significantly increased portal infiltration by macrophages (P=0.007). Taken together these results support the involvement of humorally mediated mechanisms in some cases of ACR.

Molecular case report: IgVH analysis in acute humoral and cellular liver allograft rejection suggests a selected accumulation of effector B cells and plasma cells.

Acute cellular (CLR) and humoral liver allograft rejection (HLR) are the most important immunological obstacles to successful liver transplantation. In HLR, serum antibodies play the central pathogenetic role. In CLR, CD3+ T lymphocytes drive the destructive immune response. Although CLR and HLR show different clinical symptoms and can be kept apart in most cases, they share histomorphological similarities. In CLR, hepatic B lymphocytes and plasma cells as well as B-cell-activating cytokines have recently been described, indicating that, in addition to T cells, antibody-mediated mechanisms might be involved. To analyze the impact of hepatic B cells in CLR and HLR, the immunoglobulin (Ig) variable (V)-region gene repertoire was determined from tissue of one case of CLR and one case of HLR. Complement deposits and lymphocytic infiltrate were determined using immunohistochemistry. T cells, B lymphocytes and plasma cells could be detected in both cases, whereas C3c and C4d deposits could only be demonstrated in the HLR case. The molecular analysis of 63 V-region genes showed that B cells in both allografts expressed selected V-gene repertoires. All sequences differed from the putative germline sequences by multiple somatic mutations. This suggests a clonal expansion of selected effector B cells in the portal tracts of liver allografts. Locally accumulated B cells and their antibodies might be involved in IgG-mediated complement activation in CLR and HLR.

Aberrant right subclavian artery as a new cardiac sign in second- and third-trimester fetuses with Down syndrome.

OBJECTIVE: The right subclavian artery arises normally as the first vessel from the brachiocephalic artery of the aortic arch. An aberrant right subclavian artery arises as a separate vessel from the aortic isthmus and crosses to the right, behind the trachea. This variant is present in <1% of the normal population; however, in subjects with Down syndrome, an incidence between 19% and 36% was reported. The purpose of this study was to assess the possibility of the detection of an aberrant right subclavian artery in fetuses with Down syndrome. STUDY DESIGN: Fourteen consecutive fetuses with prenatally detected Down syndrome were examined between 18 and 33 weeks of gestation. The presence of an aberrant right subclavian artery was determined by visualization of the transverse 3-vessel trachea view of the upper thorax with color Doppler ultrasonography. RESULTS: The right subclavian artery was visualized in 100% of fetuses (14/14) with Down syndrome. An aberrant right subclavian artery was identified in 35.7% of trisomy 21 fetuses (5/14). In 1 fetus, the aberrant right subclavian artery was the only abnormal ultrasound finding. In 3 fetuses, an aberrant right subclavian artery was associated with an intracardiac echogenic focus plus additional extracardiac markers. In the fourth fetus, an aberrant right subclavian artery was associated with an atrioventricular septal defect. All 9 fetuses with Down syndrome with a normal origin of the right subclavian artery had additional cardiac and/or extracardiac abnormalities. In 12 cases, pregnancy was terminated; 2 fetuses were live born. CONCLUSION: This preliminary study suggests that the in utero identification of an aberrant right subclavian artery may be a new ultrasound marker to be found in fetuses with Down syndrome. Further studies are required to assess the incidence of aberrant right subclavian artery in normal fetuses.