Free surface flow between two horizontal concentric cylinders.

Results are reported on a combined experimental and numerical investigation of a free surface flow at small Reynolds numbers. The flow is driven by the rotation of the inner of two horizontal concentric cylinders, with an inner to outer radius ratio of 0.43. The outer cylinder is stationary. The annular gap is partially filled, from 0.5 to 0.95 full, with a viscous liquid leaving a free surface. When the fraction of the annular volume filled by liquid is 0.5, a thin liquid film covers the rotating inner cylinder and reenters the liquid pool. For relatively low rotation speeds, the evolution of the film thickness is consistent with the theory for a plate being withdrawn from an infinite liquid pool. The overall liquid flow pattern at this condition consists of two counter-rotating cells: one is around the inner cylinder and the other with weaker circulation rate is in the bottom part of the annulus and nearly symmetric about the vertical axis. With increasing rotation rate, the free surface becomes more deformed, and the dynamics of the stagnation line and the cusp line dividing the cells are tracked as quantitative measures of the interface shape. In addition, the recirculating flow cells lose symmetry and the cusp deforms the free surface severely. A comparison of numerically computed flow which describes the interface by a phase-field method confirms the dynamics of the two cells and the interface deformation. For filling fraction 0.75, the liquid level is slightly above the inner cylinder and a significant decrease in size of the bottom cell with increasing rotation rate is found. For filling fractions approaching unity, the liquid flow consists of one single cell and the surface deformation remains small.

The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.

AIMS/HYPOTHESIS: Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. METHODS: LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. RESULTS: LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from >or=90% (<10 years) to >or=97% (>20 years) and remained constant thereafter. CONCLUSIONS/INTERPRETATION: Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.

Gerota and Brâncusi: Romanian anatomy and art face to face.

Gerota's name is associated with two eponyms and two histochemical methods, but he was also Brâncusi's teacher and supervisor. When Brâncusi was a student in Bucharest, he produced an 'écorché' (flayed man), of which six plaster replicas still exist, two in apparently the original shape and four which have been modified/'cosmetized'. The two are in the University of Arts in Bucharest, one in the main hall, the other in the classroom used for teaching students. Of the other four, one is in the Museum of Arts and one in the Museum of Natural Sciences of Carol I National College in Craiova (where Gerota graduated in 1885), and one each in the Faculties of Medicine in Cluj Napoca and Iassy. One more probably existed in the Faculty of Medicine in Bucharest, and one in the Faculty of Fine Arts in Iassy but no trace of them could be found. The original clay model was lost (or destroyed during transportation) in the 1930s or 1956. Two variants of the écorché exist: one 'artistic' (slender and smoother) in the University of Arts in Bucharest; the other more 'anatomical' (muscular, robust, athletic) in Craiova, Cluj and Iassy. Both variants are a very realistic representation of the human muscular system, but with that extra which only a master artist can add. Interestingly, the head of the écorché bears a striking resemblance in attitude and curves to that of Brâncusi's famous head of Mademoiselle Pogany. The replicas appear to have been distributed to embellish the capitals of four of the six historical Romanian provinces: Muntenia (Bucharest), Oltenia (Craiova), Moldavia (Iassy), and Transylvania (Cluj).

Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence.

The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein. However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated. A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli. The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides. The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli. A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity. These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.

Transcriptional repression mediated by the WT1 Wilms tumor gene product.

The wt1 gene, a putative tumor suppressor gene located at the Wilms tumor (WT) locus on chromosome 11p13, encodes a zinc finger-containing protein that binds to the same DNA sequence as EGR-1, a mitogen-inducible immediate-early gene product that activates transcription. The transcriptional regulatory potential of WT1 has not been demonstrated. In transient transfection assays, the WT1 protein functioned as a repressor of transcription when bound to the EGR-1 site. The repression function was mapped to the glutamine- and proline-rich NH2-terminus of WT1; fusion of this domain to the zinc finger region of EGR-1 converted EGR-1 into a transcriptional repressor.

Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides.

The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.

Thyrotrophin-releasing hormone, vasoactive intestinal peptide, prolactin-releasing peptide and dopamine regulation of prolactin secretion by different lactotroph morphological subtypes in the rat.

In the male rat anterior pituitary, three morphological subtypes of cells secreting primarily prolactin (PRL) (lactotrophs) have been described. Type I contain predominantly large irregularly shaped granules, whereas type II and type III lactotrophs contain smaller spherical granules. We have previously shown that oestradiol and testosterone exert a rapid stimulatory effect selectively on type II lactotrophs but it is not known how the lactotroph subtypes respond to peptide secretagogues. We have therefore examined which cell subtype(s) release PRL in response to vasoactive intestinal peptide (VIP), thyrotrophin-releasing hormone (TRH) and prolactin-releasing peptide (PrRP-31). Pituitary segments were incubated in medium containing tannic acid (to capture exocytosis of secretory granules), either alone or with secretagogue peptide. VIP (1-10 nM), TRH (10 nM) and PrRP-31 (10 nM) all caused a significant increase (P < 0.05) in the amount of PRL granule exocytosis from type II and III lactotrophs, but had no effect on PRL exocytosis from type I. Dopamine (100 nM) inhibited basal exocytosis of immunoreactive (ir)-PRL from type I, II and III lactotrophs and PrRP-31-stimulated ir-PRL granule exocytosis from II and III lactotrophs. Treatment of lactating female rats with the dopamine D(2) receptor antagonist sulpiride (40 microg/kg) produced a significant increase (P < 0.05) in PRL granule exocytosis from type I and type III lactotrophs and a significant increase (P < 0.05) in the proportion of type I and II cells undergoing exocytosis of PRL. In conclusion, VIP, TRH and PrRP-31 selectively stimulate exocytosis from type II and III lactotrophs in the male rat, whereas all three lactotroph types are sensitive to dopamine inhibition of exocytosis in male and female rats.

Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis.

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.

Parameters influencing intestinal epithelial permeability and microparticle uptake in vitro.

The hypothesis that, in vivo in situ, villous uptake of 2 microm latex microparticles involves changes at enterocyte tight junctions (TJs) was tested using Caco-2 cells on porous membranes. Epithelial permeability was measured by transepithelial resistance (TER) and particle numbers in surface, intraepithelial and sub-epithelial compartments by microscopy. Apical particle or medium addition initially closed TJs, but this was subsequently reversed in particle-treated groups. Peristaltic onward movement of a bolus was simulated by removing apical particles after an exposure period and leaving the remaining particles to interact with the epithelium: this produced marked TJ loosening during the interaction period. For particle exposure groups, the early similarity with particle numbers in vivo taken up in young adult rats became less marked with time, although bolus removal counteracted this tendency. The TJ response to vasoactive intestinal polypeptide (VIP) was time-dependent. Adsorbed and intraepithelial particle numbers increased with particle exposure time; epithelial-associated microparticle aggregation varied with treatment and submembranous particles were seen in all groups. Correlation between TER changes and particle numbers suggests TJ loosening may be important in microparticle uptake. This Caco-2 model gives epithelial particle numbers that approximate well to published figures for microparticle uptake in vivo and allows effective microenvironmental manipulation.

Multistate Outbreak of Human Salmonella Typhimurium Infections Linked to Pet Hedgehogs - United States, 2011-2013.

Zoonotic Salmonella infections cause approximately 130 000 illnesses annually in the United States. Of 72.9 million US households owning at least one pet, five million own small mammals; 3000 hedgehogs were documented by USDA in USDA-licensed breeding facilities and pet stores in 2012. State health department collaborators and PulseNet, the national bacterial subtyping network, identified human infections of a Salmonella Typhimurium outbreak strain, which were investigated by CDC, USDA-APHIS and state public and animal health officials. A case was defined as an illness in a person infected with the outbreak strain identified between 1 December 2011 and 3 June 2013. Investigators collected information on patient exposures, cultured animal and environmental specimens for Salmonella, and conducted traceback investigations of USDA-licensed hedgehog facilities. There were 26 cases in 12 states. Illness onset dates ranged from 26 December 2011 to 8 April 2013. The median patient age was 15 years (range = <1-91 years); 58% were female. Among 23 persons with available information, 8 (35%) were hospitalized and one outbreak strain-associated death was reported. Of 25 patients with available information, 20 (80%) reported pet hedgehog contact in the week before illness onset. The outbreak strain was isolated from animal and environmental samples collected from three ill persons' homes in three states. Hedgehogs were purchased in geographically distant states from USDA-licensed breeders (10/17, 59%); a USDA-licensed pet store (1/17, 6%); unlicensed or unknown status breeders (3/17, 18%); and private individuals (3/17, 18%). Traceback investigations of USDA-licensed facilities did not reveal a single source of infection. Public and animal health collaboration linked pet hedgehog contact to human infections of Salmonella Typhimurium, highlighting the importance of a One Health investigative approach to zoonotic salmonellosis outbreaks. More efforts are needed to increase awareness among multiple stakeholders on the risk of illness associated with pet hedgehogs.

Perinatal glucocorticoid treatment disrupts the hypothalamo-lactotroph axis in adult female, but not male, rats.

This study aimed to test the hypothesis that the tuberoinfundibular dopaminergic neurons of the arcuate nucleus and/or the lactotroph cells of the anterior pituitary gland are key targets for the programming effects of perinatal glucocorticoids (GCs). Dexamethasone was administered noninvasively to fetal or neonatal rats via the mothers' drinking water (1 mug/ml) on embryonic d 16-19 or neonatal d 1-7, and control animals received normal drinking water. At 68 d of age, the numbers of tyrosine hydroxylase-positive (TH+) cells in the arcuate nucleus and morphometric parameters of pituitary lactotrophs were analyzed. In control animals, striking sex differences in TH+ cell numbers, lactotroph cell size, and pituitary prolactin content were observed. Both pre- and neonatal GC treatment regimens were without effect in adult male rats, but in females, the overriding effect was to abolish the sex differences by reducing arcuate TH+ cell numbers (pre- and neonatal treatments) and reducing lactotroph cell size and pituitary prolactin content (prenatal treatment only) without changing lactotroph cell numbers. Changes in circulating prolactin levels represented a net effect of hypothalamic and pituitary alterations that exhibited independent critical windows of susceptibility to perinatal GC treatments. The dopaminergic neurons of the hypothalamic periventricular nucleus and the pituitary somatotroph populations were not significantly affected by either treatment regimen in either sex. These data show that the adult female hypothalamo-lactotroph axis is profoundly affected by perinatal exposure to GCs, which disrupts the tonic inhibitory tuberoinfundibular dopaminergic pathway and changes lactotroph morphology and prolactin levels in the pituitary and circulation. These findings provide new evidence for a long-term disruption in prolactin-dependent homeostasis in females, but not males, after inappropriate GC exposure in perinatal life.

Time-specific effects of perinatal glucocorticoid treatment on anterior pituitary morphology, annexin 1 expression and adrenocorticotrophic hormone secretion in the adult female rat.

Perinatal glucocorticoid (GC) treatment is increasingly associated with long-term disturbances in hypothalamo-pituitary-adrenocortical function. In the male rat, such treatment induces profound molecular, morphological and functional changes in the anterior pituitary gland at adulthood. To determine whether these effects are sex-specific, we have examined the effects of perinatal dexamethasone treatment on the female pituitary gland, focusing on (i) the integrity of the annexin 1 (ANXA1) dependent regulatory effects of GCs on adrenocorticotrophic hormone (ACTH) release and (ii) corticotroph and folliculo-stellate (FS) cell morphology. Dexamethasone was given to pregnant (gestational days 16-19) or lactating (days 1-7 post partum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the female offspring was examined ex vivo at adulthood (60-90 days). Both treatment regimes reduced the intracellular and cell surface ANXA1 expression, as determined by western blot analysis and quantitative immunogold electron microscopic histochemistry. In addition, they compromised the ability of dexamethasone to suppress the evoked release of ACTH from the excised tissue in vitro, a process which requires the translocation of ANXA1 from the cytoplasm to the cell surface of FS cells. Although neither treatment regime affected the number of FS cells or corticotrophs, both altered the subcellular morphology of these cells. Thus, prenatal dexamethasone treatment increased while neonatal treatment decreased FS cell size and cytoplasmic area. By contrast, corticotroph size was unaffected by either treatment, as also was the size of the secretory granules. Corticotroph granule density and margination were, however, increased markedly by the prenatal treatment, while the neonatal treatment had no effect on granule density but decreased granule margination. Thus, perinatal dexamethasone treatment exerts long-term effects on the female pituitary gland, altering gene expression, cell morphology and the ANXA1-dependent GC regulation of ACTH secretion. The changes are similar but not identical to those reported in the male.

Lack of annexin 1 results in an increase in corticotroph number in male but not female mice.

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the pituitary. We have examined corticotrophs in wild-type and ANXA1 knockout mice to determine the effects of lack of ANXA1 in male and female animals. Anterior pituitary tissue from ANXA1 wild-type, heterozygote and null mice was fixed and examined (i) by confocal immunocytochemistry to determine the number of corticotrophs and (ii) by electron microscopy to examine the size, secretory granule population and secretory machinery of corticotrophs. No differences in these parameters were detected in female mice. In male ANXA1 null mice, there were approximately four-fold more corticotrophs than in wild-type animals. However, the corticotrophs in ANXA1 null mice were smaller and had reduced numbers of secretory granules (the reduction in granules paralleled the reduction in cell size). No differences in the numerical density of folliculo-stellate, gonadotroph, lactotroph or somatotroph cells were detected in male ANXA1 null mice. Plasma corticosterone, adrenocorticotrophic hormone (ACTH) and pituitary pro-opiomelanocortin mRNA were unchanged but pituitary ACTH content was increased in male ANXA1 null mice. Interleukin (IL)-6 pituitary content was significantly elevated in male and reduced in female ANXA1 null mice compared to wild-type. In conclusion, these data indicate that ANXA1 deficiency is associated with gender-specific changes in corticotroph number and structure, via direct actions of ANXA1 and/or indirect changes in factors such as IL-6.

Characterization of the zinc finger protein encoded by the WT1 Wilms' tumor locus.

We analysed the biochemical properties of the transcription factor encoded by the putative tumor-suppressor gene present at the WT1 Wilms' tumor locus. A gene containing the full-length amino acid coding sequence of human wt1 was reconstructed from synthetic oligonucleotides and cloned into expression vectors for in vitro and in vivo protein synthesis. Polyclonal rabbit antibodies specific for the WT1 protein were raised to an Escherichia coli-produced 91 amino acid N-terminal segment and to a 136 amino acid C-terminal segment, which contains the zinc finger domain. WT1 produced by in vitro translation migrated as a 52 kDa protein on sodium dodecylsulfate-polyacrylamide gels and bound to the EGR consensus sequence in gel-retardation assays. Expression of the wt1 gene via transient transfection in COS-1 cells revealed a 52 kDa protein which was immunoprecipitated by both the N-terminal- and C-terminal-specific antisera. Immunofluorescence studies of wt1-transfected COS-1 cells revealed that the WT1 protein was localized to the nucleus. Metabolic labeling with [32P]orthophosphate failed to reveal significant phosphorylation of the WT1 protein in COS-1 cells. Two immunoreactive WT polypeptides of 52 and 54 kDa were observed in murine embryonic stem cells and COS-1 kidney cells and may represent previously identified splicing variants of WT1. These antisera should be useful in characterizing the structure and function of the WT1 protein in human Wilms' tumor specimens.

Perinatal glucocorticoid treatment produces molecular, functional, and morphological changes in the anterior pituitary gland of the adult male rat.

Stress or glucocorticoid (GC) treatment in perinatal life can induce long-term changes in the sensitivity of the hypothalamo-pituitary-adrenocortical axis to the feedback actions of GCs and, hence, in GC secretion. These changes have been ascribed largely to changes in the sensitivity of the limbic system, and possibly the hypothalamus, to GCs. Surprisingly, the possibility that early life stress/GC treatment may also exert irreversible effects at the pituitary level has scarcely been addressed. Accordingly, we have examined the effects of pre- and neonatal dexamethasone treatment on the adult male pituitary gland, focusing on the following: 1) the integrity of the acute annexin 1 (ANXA1)-dependent inhibitory actions of GCs on ACTH secretion, a process requiring ANXA1 release from folliculostellate (FS) cells; and 2) the morphology of FS cells and corticotrophs. Dexamethasone was given to pregnant (d 16-19) or lactating (d 1-7 postpartum) rats via the drinking water (1 microg/ml); controls received normal drinking water. Pituitary tissue from the offspring was examined ex vivo at d 90. Both treatment regimens reduced ANXA1 expression, as assessed by Western blotting and quantitative immunogold labeling. In particular, the amount of ANXA1 located on the outer surface of the FS cells was reduced. By contrast, IL-6 expression was increased, particularly by the prenatal treatment. Pituitary tissue from untreated control rats responded to dexamethasone with an increase in cell surface ANXA1 and a reduction in forskolin-induced ACTH release. In contrast, pituitary tissue from rats treated prenatally or neonatally with dexamethasone was unresponsive to the steroid, although, like control tissue, it responded readily to ANXA1, which readily inhibited forskolin-driven ACTH release. Prenatal dexamethasone treatment reduced the size but not the number of FS cells. It also caused a marked reduction in corticotroph number and impaired granule margination without affecting other aspects of corticotroph morphology. Similar but less marked effects on pituitary cell morphology and number were evident in tissue from neonatally treated rats. Our study shows that, when administered by a noninvasive process, perinatal GC treatment exerts profound effects on the adult pituitary gland, impairing the ANXA1-dependent GC regulation of ACTH release and altering the cell profile and morphology.

Human islet amyloid polypeptide accumulates at similar sites in islets of transgenic mice and humans.

The cellular mechanisms responsible for conversion of islet amyloid polypeptide (IAPP) into insoluble amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM) are not clear. Overexpression of IAPP and the amino acid sequence of human IAPP (hIAPP) have both been implicated. To examine factors involved in amyloid formation, transgenic mice expressing the hIAPP or rat IAPP (rIAPP) gene were generated. These mice had elevated plasma IAPP concentrations, and they were normoglycemic and normoinsulinemic. No amyloid deposits were detected by light microscopy. To examine the ultrastructure of islets, pancreatic tissue was studied from hIAPP and rIAPP transgenic mice and from age-matched control mice by immunoelectron microscopy. IAPP was immunolocalized in beta-cell secretory granules of all mice, and the COOH- and NH2-terminal flanking peptides of hIAPP were localized in beta-cell granules of hIAPP mice. Accumulations of nonfibrillar perivascular IAPP-immunoreactive material were found between capillaries and beta-cells in hIAPP transgenic mice but not in rIAPP transgenic or control mice. Similar nonfibrillar masses were identified in islets of an NIDDM patient. Secondary lysosomes in beta-cells and macrophages of hIAPP transgenic mice showed dense labeling for IAPP. We suggest that hIAPP is degraded more slowly than rIAPP or mouse IAPP by beta-cell lysosomes. Accumulations of IAPP in islet perivascular spaces may represent the early stages of islet amyloid formation.

Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.

Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.

Immunologic protection afforded by sunscreens in vitro.

Several studies have suggested a lack of correlation between sunscreen sun protection factor and protection of the skin immune system, potentially allowing greater damage to the skin by removing the natural protective erythemal response to sun exposure. Despite this, routine testing of immune protection afforded by sunscreens is not performed by industry. Current laboratory methods for investigating the efficacy of sunscreen protection of epidermal immune function use the induction of contact hypersensitivity or epidermal cell alloantigen presentation. Animal models, cell culture systems, and in vivo human studies are commonly employed, but all these systems have significant drawbacks for use in routine testing. The purpose of this study was to develop an in vitro system for testing the immunologic protection afforded by sunscreens in human skin. Five test sunscreens plus a vehicle control were tested in a "blind" fashion for their in vitro level of immune protection. Creams were applied in a standard manner to human whole skin explants and were irradiated over a range of physiologic doses using an Oriel solar simulator. A mixed epidermal lymphocyte reaction was used to quantify epidermal alloantigen-presenting capacity, in the presence or absence of test cream, for five explants. Results consistently demonstrated that all the test sunscreens protected beyond their designated sun protection factors, whereas the vehicle conferred no protection. The explant-mixed epidermal lymphocyte reaction system gave consistent, reproducible results and may prove useful for the allocation of an immune protection factor to all sunscreens.

Diagnostic and therapeutic evaluation of an anti-Langerhans cell histiocytosis monoclonal antibody (NA1/34) in a new xenograft model.

Scintigraphy using monoclonal antibodies has been suggested as a possible adjunct to conventional staging techniques for the routine staging and diagnosis of Langerhans cell histiocytosis. In this study we have developed a model for Langerhans cell histiocytosis comprising a CD1a-positive subcutaneous xenograft in the flanks of nude (nu/nu) mice. The anti-CD1a murine monoclonal antibody NA1/34 was investigated for its potential both as an imaging and as a therapeutic targeting agent in this model. Biodistribution with NA1/34 compared with irrelevant isotype-matched monoclonal antibody demonstrated specific accumulation within the xenografts of 10.0%id per g (percentage injected dose per gram) and 3.3%id per g at 48 h postinjection, respectively. NA1/34 displayed no specific accumulation to CD1a-negative xenografts. F(ab')2 fragments of NA1/34 displayed a faster clearance time of 19.6 h compared with the intact antibody, 122.4 h, resulting in a more rapid maximum xenograft uptake time of 5 h compared with 48 h postinjection for the intact antibody. Although the overall xenograft/tissue ratio for the F(ab')2 was at no time greater than that for the intact antibody, the F(ab')2 did display dramatically greater xenograft/blood ratios, reaching 19:1 at 120 h postinjection Xenograft regression using single doses of 350 microCi and 500 microCi 131I-labeled NA1/34 significantly (p < 0.001) delayed xenograft progression compared with control nonirradiated xenografts, with average delays of 3.2 and 5.7 times the control, respectively. This study suggests that the anti-CD1a monoclonal antibody, NA1/34, offers advantages in the prognosis and staging of Langerhans cell histiocytosis, in a human setting. We discuss the advantages of radioimmunoscintigraphy over conventional differential diagnostic techniques. The potential for the future radioimmunotherapy of Langerhans cell histiocytosis is also discussed.