The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress.

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.

Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737.

Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.

Isolation of clonogenic, long-term self renewing embryonic renal stem cells.

A tissue stem cell should exhibit long-term self-renewal, clonogenicity and a capacity to differentiate into the tissue of origin. Such a postnatal renal stem cell has not been formally identified. The metanephric mesenchyme (MM) of the developing kidney gives rise to both the renal interstitium and the nephrons and is regarded as the progenitor population of the developing kidney. However, isolated MM does not self renew and requires immortalization for survival in culture. Here we report the isolation and sustained culture of long-term repopulating, clonal progenitors from the embryonic kidney as free floating nephrospheres. Such cells displayed clonal self renewal for in excess of twenty passages when cultured with bFGF and thrombin, showed broad mesodermal multipotentiality, but retained expression of key renal transcription factors (Wt1, Sall1, Eya1, Six1, Six2, Osr1 and Hoxa11). While these cells did display limited capacity to contribute to developing embryonic kidney explants, nephrospheres did not display in vitro renal epithelial capacity. Nephrospheres could be cultured from both Sall1(+) and Sall1(-) fractions of embryonic kidney, suggesting that they were derived from the MM as a whole and not specifically the MM-derived cap mesenchyme committed to nephron formation. This embryonic renal stem cell population was not able to be isolated from postnatal kidney confirming that while the embryonic MM represents a mulitpotent stem cell population, this does not persist after birth.

Soft tissue sarcomas of the head and neck: a single-centre experience.

OBJECTIVE: The aim of this study was to report our experience with malignant and borderline soft tissue tumours (STS) of the head and neck region in the period 1977-2000. DESIGN: Retrospective case study including new evaluation of histological specimens. SETTING: Tertiary centre, single centre. PARTICIPANTS: Review of patient's records and new evaluation of pathological specimens were made for 66 patients. After evaluation only 36 patients (26 men and 10 women) still met present criteria for a STS in head and neck in adults. RESULTS: The original histological diagnosis was changed in 27 (41%) of the 66 patients with a primary diagnosis of sarcoma. After review the most common histological diagnoses were leiomyosarcoma (5) and rhabdomyosarcoma (5). Overall 5-year survival rate was 60%. Overall 5-year disease-free survival rate was 44%. The study showed that both tumour grade and surgical margin had a statistically significant impact on survival. No relation was found between survival and tumour size or age. CONCLUSION: The grave prognosis especially for high-grade tumours emphasizes the need for improved treatment strategies. Furthermore, conclusions from older studies concerning prognosis may be obsolete as approximately 40% of tumours previously diagnosed as sarcomas may be invalid by present day standards.

Filter-grown TR146 cells as an in vitro model of human buccal epithelial permeability.

Use of a cell culture model of a specific epithelium requires documentation of its differentiation. This study reports permeability of mannitol concurrent with a profile of differentiation markers of filter-grown TR146 cells, a cell line originating from a human buccal carcinoma, cultivated submerged or at the air-liquid interface for 23 to 31 d. A multilayered squamous epithelial-like tissue was found. The maximal permeability barrier and the most distinct stratification were obtained at day 23, when cultured submerged (apparent permeability coefficient 4.08 +/- 0.15 (x 10(-6)) cm/s; transepithelial electrical resistance 102 +/- 5 omega x cm2). The profile of differentiation markers demonstrated similarities to normal human buccal epithelium by expression of K4, K10, K13, K16, and K19 keratins, plasma membrane-associated transglutaminase, involucrin, and epidermal growth factor receptor. Uniform expression of K5, K8 and K18 was consistent with the carcinogenic origin of TR146 cells. Identical profiles of differentiation markers were obtained irrespective of method or time of culture. Karyotyping proved the human origin of TR146 cells. Three different passages had near triploid (3n+-) chromosome compliments and consistent occurrence of four marker chromosomes [mar4, mar5, mar9, and add(5)(p)], while differences between them defined them as subclones. The results indicate that a submerged filter-grown TR146 cell culture at day 23 of culture has the potential to model the human buccal epithelial barrier for permeation of drugs.

The EGF receptor system in head and neck carcinomas and normal tissues. Immunohistochemical and quantitative studies.

The EGF receptor (EGF receptor) and two of the ligands, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF), exert mitogenic activities in epithelial cells. Hence, the overall aim of this work was delineation of the EGF receptor system in head and neck carcinomas, which in the majority of cases are epithelial derived tumours. Chapter 1 is a general introduction to head and neck carcinomas and the relevance of the EGF receptor system in this context. Chapter 2 focuses on the immunohistochemical distribution of TGF-alpha in normal human tissues, while previous studies dealing with the growth factor in head and neck carcinomas revealed other localizations in normal cells. TGF-alpha was detected with monoclonal as well as polyclonal antibodies. The results showed that the growth factor is widely distributed in normal human tissues and thus not limited to malignant cells. Chapter 3 describes the immunohistochemical localization of the EGF receptor in 55 patients with squamous cell carcinoma in the head and neck region. The study included adjacent normal mucosa and in 12 cases additional dysplastic areas were present. The EGF receptor was found in the basal cell layer in normal oral and laryngeal mucosa. In sections from patients who had received preoperative irradiation the receptor was in addition seen on the spinous cells. In dysplastic epithelial all cells stained for the EGF receptor. The majority of the head and neck carcinomas expressed the EGF receptor. In poorly differentiated tumours almost all cells were positive for the receptor. Sections from moderately and well differentiated tumours demonstrated a reduction in the extent of stained areas, paralleling the situation observed in the differentiated upper layers of normal oral and laryngeal mucosa. Furthermore, this chapter describes the EGF receptor quantitatively in 60 patients with head and neck carcinoma. This study was performed in order to evaluate if overexpression of the EGF receptor was a common motif for head and neck carcinomas. The level in tumour biopsies was compared with the level in the patients' corresponding normal mucosa. An enzyme-linked immunosorbent assay detecting protein epitopes of the receptor was employed. Overexpression of the receptor was found in the majority of cases. The overexpression was further correlated to clinicopathological parameters. However, no significant correlations were found although the mean values increased with increased tumour size and advanced clinical stage. The use of quantitative assays are further discussed and limitations are emphasized with respect to heterogeneity at the EGF receptor level and the varying stromal components in malignant tissues. Despite these problems the relevance of the EGF receptor a therapeutic situation is illustrated with e.g. EGF receptor antibodies and tyrosine-kinase inhibitors. Chapter 4 focuses on the immunohistochemical expression of EGF and TGF-alpha in carcinomas from same 55 patients. This study included adjacent normal mucosa in which the growth factors were expressed above the basal cell layer. The majority of the tumours expressed both growth factors and none of the sections were negative for both EGF and TGF-alpha. In biopsies from moderately and well differentiated tumours the growth factors were demonstrated in the more differentiated cells. However, in poorly differentiated tumours the cells were positive for EGF and TGF-alpha. Chapter 5 describes immunohistochemical and quantitative changes of salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer. This study was initiated because irradiated oral and laryngeal mucosa have demonstrated staining for the receptor in the basal cell layer as well as in the spinous cells, indicating an upregulation of the receptor in response to lack of EGF. In normal biopsies from the glandula submandibularis and glandula parotis, EGF and amylase were demonstrated in the serous acini, whereas haptoc

Immunolocalization of transforming growth factor alpha in normal human tissues.

Transforming growth factor alpha (TGF-alpha) is a polypeptide with well-characterized growth promoting properties. The effects are exerted through the epidermal growth factor receptor (EGF receptor), which is present on many different kinds of cells. The growth factor was initially shown to induce anchorage-independent growth of normal cells and was, therefore, considered as an "oncogenic" growth factor. Later, its immunohistochemical presence in normal human cells as well as its biological effects in normal human tissues have been demonstrated. The aim of the present investigation was to elucidate the distribution of the growth factor in a broad spectrum of normal human tissues. Indirect immunoenzymatic staining methods were used. The polypeptide was detected with a polyclonal as well as a monoclonal antibody. The polyclonal and monoclonal antibodies demonstrated almost identical immunoreactivity. TGF-alpha was found to be widely distributed in cells of normal human tissues derived from all three germ layers, most often in differentiated cells. In epithelial cells, three different kinds of staining patterns were observed, either diffuse cytoplasmic, cytoplasmic in the basal parts of the cells, or distinctly localized adjacent to the nucleus, usually on the luminal aspect, corresponding to the localization of the Golgi complex. The latter staining pattern was seen predominantly in secretory epithelial cells. The present study thus confirms previous studies and elaborates new localizations of TGF-alpha in normal human tissues by investigating a broad spectrum of tissues in detail.

Immunohistochemical and quantitative changes in salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer.

Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in the salivary secretion of EGF are of interest because of the importance of EGF in mucosal regeneration. Immunohistochemical studies on normal tissue from parotid and submandibular glands have demonstrated EGF in the serous acini with a tendency to single cell expression in the parotid gland. Amylase has been found in the serous acini of both the submandibular and parotid glands. Haptocorrin was localized in the duct system of both glands. In the submandibular glands with radiotherapy induced sialoadenitis only very few acini with weak or no staining for EGF and amylase were demonstrated, while no changes were observed in the staining for haptocorrin. Analysis on stimulated whole saliva samples collected from 20 healthy individuals and from 20 patients prior to, and 1, 2 and 3 weeks following radiotherapy showed significant reduction in salivary contents of EGF and amylase after treatment as expressed per g protein (p < 0.0002). The salivary content of haptocorrin increased significantly after treatment (p < 0.002). These alterations may be explained by the different cellular sites of the molecules studied, the serous acini being more sensitive to ionising radiation than the duct system. The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p < 0.02), which may indicate that the tumors induce increased secretion of salivary EGF, or alternatively that the oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.

A sensitive enzyme-linked immunosorbent assay used for quantitation of epidermal growth factor receptor protein in head and neck carcinomas: evaluation, interpretations and limitations.

The EGF receptor is a transmembrane glycoprotein exerting mitogenic effects on epithelial cells. The purpose of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for determination of the epidermal growth factor receptor (EGFR) protein to examine whether the receptor was overexpressed in head and neck squamous cell carcinomas compared with the normal counterpart, and to establish whether clinicopathological correlations were present by investigating a broad spectrum of parameters (tumour size, clinical stage, positive lymph nodes, tumour site, histological grade, keratinisation, preoperative irradiation and clinical outcome). The assay employs two commercially available monoclonal antibodies, both detecting protein epitopes. The material comprises 60 head and neck carcinomas, corresponding normal tissue and normal oral mucosa from healthy individuals. The study demonstrates significantly higher receptor levels in tumours compared with normal tissue (P < 0.002) and a range in tumours and normal tissues of 0.4-10.5 and 0.1-4.3 nmol g-1 membrane protein respectively. Quantitation of receptors in normal mucosa emphasises the importance of using the patients' corresponding normal tissue, because using the patients' mucosa resulted in 83% overexpression, while using normal mucosa from healthy individuals only demonstrated overexpression in 50% of cases. No significant clinicopathological correlations could be established, although the mean values for EGFR increased with tumour size and advanced clinical stage. Furthermore, the prognostic value concerning disease-free survival, recurrence and the time interval for recurrence were investigated but no significance could be demonstrated. In conclusion, the investigation supports the theory of overexpression of EGFR protein as a common motif for malignant epithelial tumours, but limitations in interpretations are demonstrated and discussed further.

General health assessment in refugees claiming to have been tortured.

General health assessment of refugees claiming to have been previously exposed to torture takes place in a psychological atmosphere affected by the difficult situation of the refugee. Thirty-one refugees, mainly from the Middle East and Africa, were assessed as regards their physical and mental health. Assessment took place with the help of professional interpreters and was, during each interview, performed by two medical doctors using double-blind techniques. Based on a number of highly significant (P < 0.001) correlation coefficients and Kappa values, observers agreed frequently on gradients of symptom intensity and less frequently on absolute symptom levels. However, agreement was almost complete when assessing the presence of intense symptoms and the absolute absence of a symptom. Symptom patterns were demonstrated to be consistent, clinically interpretable and, furthermore, closely associated (P < 0.0001) with self-reported global (general) health. Reliability was moderate with respect to clinical observation during interview.

Transforming growth factor alpha and epidermal growth factor in laryngeal carcinomas demonstrated by immunohistochemistry.

Fifteen laryngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) using immunohistochemical methods. In a recent study the same material was characterized for epidermal growth factor receptors (EGF receptors) which were confined predominantly to the undifferentiated cells. The expression of this growth factor system in malignant cells may play a role in carcinogenesis and/or tumour growth. All carcinomas were positive for TGF-alpha and 12 were positive for EGF. In moderately-to-well differentiated carcinomas, the immunoreactivity was mainly detected in the cytologically more differentiated cells. Nine sections included both laryngeal stratified squamous epithelium of normal appearance and carcinoma. The immunoreactivity was here again localized in the cytologically more differentiated cells above the basal cell layer. The present investigation and our previous results confirm the existence of EGF receptors, TGF-alpha and EGF in laryngeal carcinomas. In addition, we conclude that the conditions do exist for growth factors to act through an autocrine system in poorly differentiated tumours and through a paracrine system in the moderately-to-well differentiated tumours.

Alterations of fibrillarin distribution and nucleolar ultrastructure induced by adenovirus infection.

Nucleolar modifications induced by adenovirus type 5 (Ad5) have been examined in HeLa cells by bismuth staining and immunogold labeling of fibrillarin, both of which visualize the dense fibrillar component (DFC) of nucleoli. A progressive increase in the compaction of the nucleolus is accompanied both by a migration of strands of DFC towards the border of the nucleolar body and by a decrease in the amount of fibrillarin within the bismuth stained DFC. In addition, the bismuth-stained extranucleolar fibrillar spots induced by Ad5 infection often were intensely labeled with monoclonal anti-fibrillarin antibody. Previously reported defects in ribosomal RNA expression during adenovirus infection could result from the migration and accumulation of nucleolar proteins implicated in ribosomal biogenesis into virus-induced extranucleolar structures.

Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas.

Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected with a monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa, the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus paralleling the situation observed in the normal differentiated oral mucosa. In four cases, material was available from both a primary tumour and a metastasis. Three of these were positive for TGF-alpha and EGF with the same staining pattern as that of the primary tumours. This investigation together with our previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases.

Mapping of monoclonal antibody epitopes in the nucleolar protein fibrillarin (B-36) of Physarum polycephalum.

We have mapped the epitopes for nine monoclonal antibodies raised against the nucleolar protein fibrillarin of the slime mold Physarum polycephalum. This has been done using a combination of specific chemical and enzymatic cleavage, Western blotting and partial sequencing of fragments. Cleavage with cyanogen bromide reveals four prominent methionine cleavage sites within the protein. Western blotting shows that none of the monoclonal antibody epitopes are dependent on long range interactions. Eight highly-conserved epitopes are clustered in the carboxy terminal half of the protein, while a single less-conserved epitope (for monoclonal antibody P1G12) is located at the amino terminus and appears to lie within the Gly/DMA/Phe domain.

Immunohistochemical detection of epidermal growth factor receptor in laryngeal squamous cell carcinomas.

Laryngeal squamous cell carcinomas from 15 consecutive preoperatively irradiated patients were investigated for the expression of epidermal growth factor receptor (EGF receptor). The study was performed on frozen sections by means of the 5-layer APAAP technique employing an antibody recognizing the extracellular part of the EGF receptor. In sections from 9 of the patients with laryngeal squamous cell carcinoma, normal differentiated epithelia were included. Sections from 6 of these patients, in addition, contained dysplastic epithelia. Expression of EGF receptor-like material was demonstrated in the basal cell layer of normally differentiated laryngeal epithelial and in dysplastic epithelia. Fourteen of the squamous cell carcinomas proved EGF receptor positive. Nearly all cells in the poorly differentiated carcinomas showed positive staining with the antibodies. In moderately to well differentiated carcinomas a reduction in the extent of staining was seen in certain areas. Especially for the epithelial pearls, the staining reaction was localized to the undifferentiated cells in the periphery. This finding corresponds to the staining pattern observed in the basal cell layers of normal epithelial. The present investigation confirms the expression of EGF receptor-like material in normal laryngeal epithelial, dysplastic epithelial and squamous cell carcinoma. The staining pattern was similar to that observed in oral squamous cell carcinomas, predominantly varying inversely with cellular differentiation.

Epidermal growth factor receptor expression on oral mucosa dysplastic epithelia and squamous cell carcinomas.

The expression of the receptor for epidermal growth factor (EGF) has been determined on oral squamous cell carcinomas. Immunoreactive receptor was localized using a monoclonal anti-EGF-receptor antibody which reacts with sequences in the external domain of the receptor. Frozen sections were studied from 40 patients with squamous cell carcinomas. In 16 sections from the patients with the squamous cell carcinomas, normal differentiated oral mucosa was included and in 7 of these the patients had received preoperative radiotherapy. Sections from 6 other patients with squamous cell carcinoma contained dysplastic epithelia. EGF-receptor-positive cells were present in the basal cell layer on normal differentiated oral mucosa. In sections from patients receiving preoperative radiotherapy the EGF-receptor-positive cells were also found in the spinous cells. In dysplastic epithelia nearly all cells stained for the receptor. The distribution and staining intensity of the EGF receptor varied in the oral squamous cell carcinomas, 36 were positive. The staining pattern in the carcinomas obtained from patients receiving preoperative radiotherapy was not altered qualitatively. Nearly all poorly differentiated cells were stained, but when the tumor was moderately to well differentiated a reduction in the extent of staining in certain areas was seen, paralleling the findings observed in the differentiated upper layers of the normal oral mucosa. This was most pronounced for the epithelial pearls, where the EGF-receptor-positive cells were localized to the undifferentiated cells in the periphery. The results of the present investigation confirm the presence of the EGF receptor on undifferentiated cells, with the extent of the staining reaction on oral squamous cell carcinomas varying inversely with cellular differentiation.

Localization of U3 RNA molecules in nucleoli of HeLa and mouse 3T3 cells by high resolution in situ hybridization.

We have examined the ultrastructural localization of U3 RNA in the nucleoli of HeLa and mouse 3T3 cells by in situ hybridization with a biotinylated U3 DNA probe and subsequent detection of hybrids with electron microscopy by direct immunogold labeling. The highest levels of signal density for U3 RNA are detected over the dense fibrillar component (DFC) of the nucleolus, including the interfaces between DFC and the enclosed fibrillar center (FC) on the one hand and DFC and the granular component (GC) on the other hand. Lower but significant signals also are observed over GC, which indicate, taking into account the high relative volume of GC in a nucleolus, that a substantial fraction of U3 RNA is present in this compartment where the more mature forms of pre-rRNA accumulate. In parallel, the localization of fibrillarin was analyzed by immunogold detection, demonstrating that fibrillarin and U3 RNA have a roughly similar distribution, although quantitative measurements reveal that the signal ratio for both molecules exhibit significant differences among the major ultrastructural components of the nucleolus.

The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins.

The amino terminal sequence of the 34 kD nucleolar protein B-36 isolated from the slime mold Physarum polycephalum has been determined. This portion of B-36 is rich in glycine, phenylalanine and the modified amino acid asymmetrical dimethylarginine (DMA) and is 65% identical to that for fibrillarin, a similar and potentially homologous 34 kD nucleolar protein from rat. The terminus of B-36 contains an interesting nine amino acid sequence, Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly, which is precisely repeated three times in the 110 kD nucleolar protein nucleolin. Similar sequences have also been reported in a yeast nucleolar protein (SSB-1) and several hnRNP proteins (rat A1 and brine shrimp GRP33). The conserved nature of this unusual sequence is suggestive of an important function which may include RNA-binding since several of these proteins share this feature.