A global representation of vitamin D status in healthy populations.

PURPOSE: This paper visualizes the available data on vitamin D status on a global map, examines the existing heterogeneities in vitamin D status and identifies research gaps. METHODS: A graphical illustration of global vitamin D status was developed based on a systematic review of the worldwide literature published between 1990 and 2011. Studies were eligible if they included samples of randomly selected males and females from the general population and assessed circulating 25-hydroxyvitamin D [25(OH)D] levels. Two different age categories were selected: children and adolescents (1-18 years) and adults (>18 years). Studies were chosen to represent a country based on a hierarchical set of criteria. RESULTS: In total, 200 studies from 46 countries met the inclusion criteria, most coming from Europe. Forty-two of these studies (21 %) were classified as representative. In children, gaps in data were identified in large parts of Africa, Central and South America, Europe, and most of the Asia/Pacific region. In adults, there was lack of information in Central America, much of South America and Africa. Large regions were identified for which the mean 25(OH)D levels were below 50 nmol/L. CONCLUSIONS: This study provides an overview of 25(OH)D levels around the globe. It reveals large gaps in information in children and adolescents and smaller but important gaps in adults. In view of the importance of vitamin D to musculoskeletal growth, development, and preservation, and of its potential importance in other tissues, we strongly encourage new research to clearly define 25(OH)D status around the world.

Regulation of the trans-activation potential of STAT5 through its DNA-binding activity and interactions with heterologous transcription factors.

Extracellular hormones, growth factors and cytokines relay their effects on the transcription of genes through the recognition of specific receptors and intracellular signalling molecules. Signal transducers and activators of transcription (STATs) have been recognized as crucial intracellular signalling molecules. The cytokine receptor-associated Janus kinases (JAKs) convert the latent monomeric form of the STAT molecules to the activated dimeric form through tyrosine phosphorylation. The dimers bind to specific DNA response elements and are able to induce transcription. This induction requires the full-length form of the STAT molecules. Negative regulatory potential is exerted by the short form of the molecule, which lacks the trans-activation domain. This short form is activated and dimerized, but dephosphorylation is impaired. The short form of STAT occupies the DNA-binding sites in a stable fashion and acts as a strong suppressor of wild-type action. Positive enhancement of STAT5 trans-activation potential is provided by the glucocorticoid receptor. Ligand activation of this receptor causes the formation of a complex with STAT5 and deviation to the STAT5 DNA-binding site. An additional regulatory loop is provided by the reactivation of the short form of STAT5 through glucocorticoid receptor association. Conversely, classical glucocorticoid-responsive genes are negatively affected by STAT5 activation.

A novel animal model for the evaluation of the efficacy of drugs directed against the ErbB2 receptor on metastasis formation.

The ErbB2 receptor tyrosine kinase is often overexpressed in human malignancies and causally involved in transformation. High levels of ErbB2 in tumor cells correlate with an unfavorable prognosis. This makes the ErbB2 receptor an interesting target for tumor therapy, and several strategies have been designed to direct drugs to ErbB2-expressing cells. We established a novel cellular model that allows preclinical evaluation of ErbB2-directed drugs in immunocompetent animals. Renal carcinoma (Renca) cells are an established tumor cell line that originated in Balb/c mice. Upon intravenous transplantation, these cells form pulmonary metastases in Balb/c mice. The transforming genetic lesions in these cells are not fully characterized, but do not seem to involve alterations in ErbB2 gene expression. We transfected Renca cells with the gene encoding the human ErbB2 receptor to provide a target structure for specific drugs and with the bacterial lacZ gene to provide a sensitive means of detection of the tumor cells in the transplanted animals. These genetically modified cells form lung metastasis and can be easily visualized on the surface of lung tissue by staining with an X-gal solution. This allows a quantitative analysis of the number of ErbB2-expressing pulmonary metastasis. We previously used these Renca cells to evaluate the efficacy of an ErbB2-specific tumor toxin on pulmonary metastases in an adjuvant and a palliative treatment setting. In both cases, we achieved a dramatic reduction of disseminated lung lesions. Here we show that even at an advanced stage of metastasis formation, the ErbB2-specific toxin is able to efficiently reduce the number of pulmonary tumors.

Systemic treatment with a recombinant erbB-2 receptor-specific tumor toxin efficiently reduces pulmonary metastases in mice injected with genetically modified carcinoma cells.

Receptor-mediated targeted tumor therapy is an important applied consequence of the studies on the genetic causes of cancer. These therapy concepts have to be evaluated in novel animal models that reflect the molecular aberrations found in human tumors. Here we introduce an animal model that allows the evaluation of drugs directed against a surface receptor that is frequently altered in primary human adenocarcinomas. Tumor toxins are polypeptides in which a tumor cell-specific recognition domain and a toxic effector domain have been joined by DNA recombination in vitro. Tumor cell recognition is contributed by a single-chain antibody domain specific for the extracellular domain of the erbB-2 receptor [scFv(FRP5)] and cytotoxicity by the enzymatically active domain of a bacterial exotoxin (exotoxin A from Pseudomonas aeruginosa). The erbB-2 receptor is overexpressed in many primary human cancer cells and is a favorable target for directed tumor therapy. The fusion protein scFv(FRP5)-exotoxin A has previously been shown to be able to efficiently and specifically kill erbB-2 receptor-expressing tumor cells. We have investigated the potential of this tumor toxin to detect and eliminate metastasizing tumor cells upon systemic administration. Murine renal carcinoma cells genetically modified with human erbB-2 receptor and bacterial beta-galactosidase genes form large pulmonary metastases when injected into the tail vein of BALB/c mice. Administration of the tumor toxin over a 10-day time period starting 1 day after tumor cell transplantation totally suppressed the formation of metastases. The treatment of animals 11 days after tumor cell transplantation, allowing the establishment of many pulmonary metastases, led to a drastic reduction in their number and size.

Cytokine receptor-independent, constitutively active variants of STAT5.

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.

Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells.

Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.

Schwannoma cells induce a tumor-cell-specific cytotoxic-T-cell response upon transplantation into syngeneic rats but escape elimination through the secretion of immunosuppressive factors.

Tumor cells often express antigens that can be recognized by the immune system. Despite induction of an immune response, the tumor cells escape their elimination. We have studied the mechanisms and factors which mediate these events in a syngeneic tumor model. NV2Cd rat schwannoma cells were transplanted into BDIX rats. After injection of 10(7) to 2 x 10(7) cells, tumors grew very slowly for 10 to 12 days. After that time, rapid growth was observed. The tumors consisted of compact areas of spindle-shaped cells with small cysts, many blood vessels and central necrotic areas. During tumor growth, the number of spleen cells and T lymphocytes increased, and cytotoxic T cells with specificity for the NV2Cd tumor cells were detected. The strong specific cellular immune response did not prevent the increase in tumor volume. We studied the biological activity of the fluid present in the cysts of the tumor. At a concentration of 1 ng to 10 microg protein per ml, the cyst fluid inhibited the proliferation of splenic T lymphocytes and B lymphocytes and of lymphoma cells, but enhanced the proliferation of NV2Cd tumor cells. The cyst fluids contain the immunosuppressive transforming growth factors (TGF)-beta1, -beta2 and -beta3, also the vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Antibodies directed against TGF-beta relieved the suppression of T-cell growth by cyst fluid, but did not influence the proliferation of NV2Cd cells. The growth-modulating factors present in the tumor cyst fluid were also detected in conditioned medium from NV2Cd cells cultured in vitro. Our data suggest that tumors can escape the cellular immune response by the production of factors that inhibit lymphocytes. They also enhance their own growth environment by secreted factors.

Intra-tumoral application of a heregulin-exotoxin-a fusion protein causes rapid tumor regression without adverse systemic or local effects.

Tumor toxins are recombinant, bifunctional proteins which comprise a tumor-cell-specific recognition domain and an enzymatic toxin domain. We have evaluated the in vivo effects of a tumor toxin that specifically recognizes the erbB-3 and erbB-4 receptors (HRG beta 1-ETA). High doses of HRG beta 1-ETA administered systemically (intracardially or intraperitoneally) caused acute liver necrosis and were lethal. The same dose of tumor toxins applied subcutaneously had no detectable histopathological effects. The anti-tumor activity of HRG beta 1-ETA was tested in nude mice with xenografts of a human breast tumor, MAXF1162. The MAXF1162 tumor grew rapidly upon s.c. implantation. Intra-tumoral application of HRG beta 1-ETA (7 times 5 micrograms over a period of 21 days) induced complete regression of tumors. At the time the treatment was terminated, no tumor cells were detectable microscopically. Evaluation of the liver of treated animals revealed no significant toxicity in the effective dose range. These experiments indicate that tumor toxins can become valuable for local tumor treatment and for reduction of tumor burden.

Functional interactions between Stat5 and the glucocorticoid receptor.

Signal transduction pathways enable extracellular signals to activate latent transcription factors in the cytoplasm of cells. Dimerization, nuclear localization and binding to specific DNA sequences result in the induction of gene transcription by these proteins. These events are necessary for the functioning of the JAK/STAT pathway and of the glucocorticoid-receptor pathway. In the former, the protein Stat5, which is a member of a family of signal transducers and activators of transcription, is activated by cytokines, hormones and growth factors. These polypeptide ligands bind at the outside of the cell to specific transmembrane receptors and activate intracellular Janus protein tyrosine kinases (JAKs) to tyrosine-phosphorylate STAT proteins; interaction with the SH2 domain of the dimerization partner then confers the ability to bind to DNA at the STAT-response element and induce transcription. In the glucocorticoid-receptor pathway, the receptor interacts with its steroid hormone ligand in the cytoplasm, undergoes an allosteric change that enables the hormone receptor complex to bind to specific DNA-response elements (glucocorticoid response elements, or GRE) and modulate transcription. Although these pathways appear to be unrelated, we show here that the glucocorticoid receptor can act as a transcriptional co-activator for Stat5 and enhance Stat5-dependent transcription. Stat5 forms a complex with the glucocorticoid receptor which binds to DNA independently of the GRE. This complex formation between Stat5 and the glucocorticoid receptor diminishes the glucocorticoid response of a GRE-containing promoter.

Targeted inhibition of tumor-cell growth by recombinant heregulin-toxin fusion proteins.

Fusion of functional domains of proteins by in vitro recombination of gene fragments can be used to generate novel anti-tumor agents. The combination of tumor-cell-recognition functions and toxic functions results in cytotoxic molecules with a high specificity for tumor cells. Human adenocarcinomas are frequently characterized by over-expression of members of the epidermal-growth-factor (EGF) receptor family (ErbB-1, 2, 3 and 4), when compared with normal cells. These tumors are particularly suited to treatment with recombinant toxins. The human heregulins (HRG) and their rat counterparts (neu differentiation factor, NDF) have been identified as ligands for these receptors. Two chimeric heregulin-toxin fusions consisting of the EGF-like receptor recognition domain of the heregulin isoforms HRG alpha and HRG beta I, and the domains II, Ib and III of the Pseudomonas exotoxin A (ETA) were constructed. HRG beta I-ETA is highly cytotoxic for the mammary carcinoma cell lines SK-BR-3 and MDA-MB-453. HRG alpha-ETA was less active than HRG beta I-ETA. The killing activity of the recombinant toxins correlated with the expression levels of ErbB-3 and/or ErbB-4 in the cell lines studied. High expression of ErbB-2 is not sufficient to confer sensitivity towards the HRG-ETA. Treatment of mice with 0.4 mg/kg/day of HRG beta I-ETA caused growth retardation of transplanted human breast tumor cells. Higher levels of HRG beta I-ETA administration resulted in acute hemorrhagic necrosis of the liver.

An activated allele of the c-erbB-2 oncogene impairs kidney and lung function and causes early death of transgenic mice.

The pathogenicity of the human c-erbB-2 oncogene was evaluated in transgenic mice. A DNA sequence comprising the promoter-enhancer region of the MMTV LTR and a constitutively activated allele of the human c-erbB-2 growth factor receptor gene was introduced into the germ line of mice. Expression of the transgene was observed in kidney, lung, mammary gland, salivary gland, Harderian gland, and in epithelial cells of the male reproductive tract. All transgenic mice expressing the c-erbB-2 receptor died within four months of birth. Histopathological analysis suggests that preneoplastic lesions in kidney and lung most likely caused organ failure and the early death of the transgenic mice. Focal dilatation and atypical proliferation of the tubular epithelial cells was found in the kidney. These hyperplastic lesions were found adjacent to normal tubules. Immunohistochemistry showed that normal renal structures were completely negative for c-erbB-2 protein expression. Atypical pseudopapillary proliferation of bronchial and bronchiolar epithelial cells narrowed the bronchial lumen in lung. Alveoli appeared normal. The expression of c-erbB-2 protein was strictly limited to the proliferating epithelial cells and not detected in normal tissue. The mammary glands of two parous mice were underdeveloped, lacking lobular-alveolar structures and were lactation deficient. Only a few ducts were interspersed in the fat pad. A virgin mouse developed a focal adenocarcinoma infiltrating the mammary fat pad. Expression of the c-erbB-2 protein was enhanced in the proliferating epithelial cells. Transgenic males were sterile. Epithelial hyperplasia and hypertrophy in the epididymis, vas deferens and seminal vesicles was found. The transgene is not uniformly expressed in the tissues where the MMTV LTR is transcriptionally active. The scattered transgene expression invariably coincides with epithelial hyperplasia.

Astrocyte culture on nitrocellulose membranes and plastic: detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

[Delta superinfection of hepatitis B in Switzerland: histology and serology of 28 patients]. / Die Delta-Koinfektion der Hepatitis B in der Schweiz: Histologie und Serologie von 28 Patienten.

A series of 610 consecutive liver biopsies collected from Jan. 1980 to Oct. 1983 in two Swiss gastro-enterological centers was studied by immunohistology for the presence of delta-antigen, HBsAg and HBcAg in frozen sections. This led to the identification of 46 tissue and 45 serum samples of 28 patients, including 5 with follow-up biopsies. All had ongoing hepatitis B (HB) with delta-superinfection, representing 13.3% of all patients with HB. The sera were studied for HBsAg, anti-HBs, anti-HBc and HBe-markers by RIA, for anti-delta by indirect immunofluorescence on delta-positive test sections and for Dane particles by immune electromicroscopy. 18 of the 21 native Swiss patients were i.v. drug users between 20 and 28 years of age (30% of all drug users in this series). The other patients were residents from Mediterranean countries (n = 5), Eastern Europe (n = 1) and Asia (n = 1). Female patients (n = 5) were seen only among i.v. drug users. The most frequent histological type of HB was chronic active (aggressive) hepatitis (CAH) (26 of 46 biopsies or 13 of 28 patients, respectively), followed by acute HB with histological signs of possible transition to chronicity (8 of 46 biopsies or 6 of 28 patients), and acute lobular HB (6 of 46 biopsies or 5 of 28 patients). Chronic persistent HB (CPH) was rare (5 of 46 biopsies, final diagnosis in 3 patients). The delta-infection was found to persist in sequential biopsies when a chronic HB was established, the longest documented period being 72 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental non-A, non-B hepatitis in chimpanzees: light, electron and immune microscopical observations.

Two chimpanzees (Pan troglodytes) were inoculated and cross-challenged with a fibrinogen and factor VIII preparation, respectively. Successful non-A, non-B (NANB) infection was documented by biphasic elevations of aminotransferases (ALT), concomitant hepatitic reactions and typical electron microscopic alterations, the most consistent being dilatation of the endoplasmic reticulum, as well as tubular and sponge-like cytoplasmic inclusions in the absence of nuclear virus-like particles. An anti-nuclear (anti-DNA) antibody of the IgM class in one of the chimpanzees simulating an antiviral antibody is described.

Chronic interstitial nephritis in Whipple's disease.

Report is given on a 68-year-old man who suffered primarily from progressive weight loss and repeated episodes of fever and arthralgia. Later, liver dysfunction and renal insufficiency developed. Liver and kidney biopsies disclosed granulomatous hepatitis and nephritis. Because of the morphologic and clinical findings, the diagnosis of Boeck's disease was made. Shortly before death, diarrhea developed. Autopsy revealed a massive systemic involvement in Whipple's disease proven by light and electron microscopy and immunofluorescence. Tuberculoid and epitheloid cell granulomas and isolated giant cells were found in addition to the biopsy findings in skeleton muscles, the small intestine, lymphnodes and bronchi. At autopsy, the kidney showed chronic interstitial nephritis. The literature of kidney involvement in Whipple's disease is reviewed. This is the first case with granulomatous interstitial nephritis and chronic renal insufficiency in an inadequately treated Whipple's disease.

An improved method for HBcAg demonstration in paraffin-embedded liver tissue.

Factors influencing the preservation of HBcAg in formalin-fixed, paraffin-embedded liver tissue have been analysed. Fixation time, choice of the embedding medium, deparaffinization and selection of anti-HBc proved to be critical variables. For optimal results, fixation in 4% buffered formalin for no longer than 24-48 h, the use of wax with a low melting point, a heating step prior to deparaffinization, and a search for anti-HBc specifically reactive for paraffinated HBcAg are recommended.

[Delta-antigen in hepatitis B]. / Delta-Antigen bei Hepatitis B.

Among 571 liver biopsies delta-Ag was found in 10 of 365 HBsAg positive patients (9 with CAH, 1 with CPH). Delta-Ag was demonstrated in nuclei and cytoplasm of liver cells in both cryostat and paraffin sections. Follow-up studies revealed persistence of delta-Ag for as long as 6 years and temporary appearance or loss of Dane particle-associated parameters without a change in the concomitant inflammation. These findings are compatible with the hypothesis that delta-Ag represents a defective viral agent which modulates, by superinfection, the typical viral expression patterns of HBV infection.

delta Antigen in hepatitis B: immunohistology of frozen and paraffin-embedded liver biopsies and relation to HBV infection.

The finding that the recently described hepatitis B (HB)-associated delta antigen (delta Ag) is preserved in pronase-treated, formalin-fixed paraffin sections allowed a combined prospective and retrospective immunohistological study of its occurrence in 571 liver biopsies. Among 116 frozen biopsies (69 HBAg seropositive, 47 HBAg seronegative) and 455 paraffin-embedded biopsies (296 HBAg seropositive, 159 HBAg-negative), delta Ag was found in 10 HBAg seropositive patients. With the exception of 1 patient with chronic persistent HB, all had chronic-active HB and none had acute HB; 5 patients were i.v. drug abusers. In follow-up biopsies, the delta Ag persisted with HBsAg for as long as 6 years. The expression of delta Ag showed similarities to the HBcAg system including nuclear localization, mixed nuclear cytoplasmic expression, and coexistence with anti-delta in blood. The findings are compatible with the hypothesis that delta Ag represents a transmissible, defective viral agent which requires HBV as a helper and may modulate, but not terminate, ongoing HBV infection.