Yes, ICantSeeYou

Making Humans More Like Monkeys - With A Natural Resistance To AIDS

I think basically what they are saying is that monkey's have DNA that gives them a natural resistance to AIDS.  They tested to see if humans had the same gene and found it but under junk DNA (DNA which was turned off sometime in our history).   So they needed a way to turn this junk on, and they found a drug to do it.

Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1

Nitya Venkataraman¹, Amy L. Cole¹, Piotr Ruchala², Alan J. Waring², Robert I. Lehrer², Olga Stuchlik³, Jan Pohl³, Alexander M. Cole^1*

1 Department of Molecular Biology and Microbiology, Burnett College of Biomedical Sciences, University of Central Florida, Orlando, Florida, United States of America, 2 Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 3 Biotechnology Core Facility Branch, DSR, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue “demidefensin” precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Author Summary

Defensins are a large family of small antimicrobial peptides that contribute to host defense against a broad spectrum of pathogens. In primates, defensins are divided into three subfamilies—alpha, beta, and theta—on the basis of their disulfide bonding pattern. Theta-defensins were the most recently identified defensin subfamily, isolated initially from white blood cells and bone marrow of rhesus monkeys. They are the only known cyclic peptides in mammals and act primarily by preventing viruses such as HIV-1 from entering cells. Whereas theta-defensin genes are intact in Old World monkeys, in humans they have a premature stop codon that prevents their expression; they thus exist as pseudogenes. In this work, we reveal that, upon correction of the premature termination codon in theta-defensin pseudogenes, human myeloid cells produce cyclic, antiviral peptides (which we have termed “retrocyclins”), indicating that the cells retain the intact machinery to make cyclic peptides. Furthermore, we exploited the ability of aminoglycoside antibiotics to read-through the premature termination codon within retrocyclin transcripts to produce functional peptides that are active against HIV-1. Given that the endogenous production of retrocyclins could also be restored in human cervicovaginal tissues, we propose that aminoglycoside-based topical microbicides might be useful in preventing sexual transmission of HIV-1.

Introduction

Nearly 33 million people are infected with HIV worldwide [1,2], and despite extensive efforts there are no effective vaccines or other countermeasures to protect against HIV transmission [3]. In our attempts to find effective anti-HIV agents, our group determined that certain synthetic θ-defensins called “retrocyclins” are potent inhibitors of HIV-1 infection [4–8]. Retrocyclins belong to a large family of antimicrobial peptides known as defensins, all of which are cationic, tri-disulfide bonded peptides that have important roles in innate host defense. On the basis of the position of the cysteines and the disulfide bonding pattern, defensins are grouped into three subfamilies: α-defensins, β-defensins, and θ-defensins [9,10].

θ-Defensins such as retrocyclin have a cyclic peptide backbone, derived from the head-to-tail-ligation of two peptides that each contributes nine amino acids to form the 18-residue mature peptide [11]. θ-Defensins are the only known cyclic peptides in mammals and were originally isolated from rhesus macaque leukocytes and bone marrow [11–13]. While θ-defensin peptides are produced in old world monkeys and orangutans, in humans they exist only as expressed pseudogenes [14]. A premature termination codon in the signal peptide portion of human retrocyclin mRNA prevents its translation. The retrocyclin gene is otherwise remarkably intact, showing 89.4% identity with rhesus θ-defensins. Its genetic information was utilized to recreate retrocyclins synthetically and confirm their activity against both X4 and R5 strains of HIV-1 [4–7].

Retrocyclins inhibit the fusion of HIV-1 Env by selectively binding to the C-terminal heptad repeat region on gp41 blocking 6-helix bundle formation [15,16]. RC-101 is a congener of retrocyclin with a single arginine to lysine substitution that retains structural and functional similarity to retrocyclin [4]. RC-101 exhibited enhanced anti-HIV-1 activity against over two dozen primary isolates from several clades [7,8], and did not induce inflammation or toxicity in organotypic models of human cervicovaginal tissue [17]. Continuous passaging of HIV-1 BaL in the presence of subinhibitory concentrations of RC-101 for 100 days induced only minimal viral resistance [18]. Given these beneficial attributes, we envisioned that restoring the endogenous expression of retrocyclins in humans would provide an effective and natural way of combating HIV-1 infection.

In the current study we restored the translation of this evolutionarily lost retrocyclin peptide by ablating the premature termination codon using site-directed mutagenesis, and analyzed whether human cells can synthesize biologically active retrocyclins. We found that promyelocytic HL60 cells stably transfected with retrocyclin constructs in which the premature termination codon was corrected could express retrocyclins. Application of the expressed retrocyclins to TZM-bl cells, PM1 cells, and peripheral blood mononuclear cells (PBMCs) conferred protection against HIV-1 infection. Moreover, mass spectrometric techniques confirmed the presence of correctly folded mature retrocyclin peptides. We also explored methods to read-through the premature termination codon within the retrocyclin pseudogene. Previous reports revealed that aminoglycoside antibiotics could suppress the termination codon of pseudogenes and disease-associated nonsense mutations [19–25]. In bacteria, aminoglycosides bind strongly to the decoding site on the 16S rRNA, thereby hindering protein synthesis [26]. However, in eukaryotes, aminoglycosides bind to the eukaryotic decoding site with low affinity and induce a low level of translational misreading, which suppresses the termination codon through the incorporation of an amino acid in its place [27]. Herein, we utilized aminoglycosides to induce translational read-through of the θ-defensin pseudogene, which restored the expression of functional anti-HIV-1 retrocyclin peptides in human cervicovaginal tissue models. Topical application of aminoglycosides to produce endogenous retrocyclins in the vaginal mucosa might soon be an effective preventative to combat sexual transmission of HIV-1.

Results and Discussion

Creation of Promyelocytic Cells Stably Transfected with Retrocyclin Constructs

θ-Defensins are formed by post-translational modification of two 12-residue gene products, each of which is processed to give a nonapeptide that contains three cysteines. The N-terminus of one nonapeptide forms a peptide bond with the C-terminus of another nonapeptide, resulting in a cyclic 18 residue peptide with three intramolecular disulfide bonds [11,14]. To determine if human cells have retained the ability to process θ-defensins, we transfected promyelocytic HL60 cells with retrocyclin constructs each encoding a nonapeptide in which the premature termination codon was replaced with a glutamine (⊗17Q).

Four types of constructs were produced: R1, R3, A1, and A3 (Figure 1). Aside from the corrected premature termination codon (⊗17Q), all constructs were engineered to contain two termination codons at the end of the gene to ensure read-fidelity. Constructs with an “R” designation terminate after the retrocyclin portion of the gene, while constructs with an “A” designation contain the retrocyclin portion with additional downstream residues that might be critical for translation and/or processing [14,28]. Constructs with a “1” designation do not have any additional residues mutated, while constructs with a “3” designation have the additional Arg → Lys mutation (R70K) encoding the RC-101 nonapeptide. HL60 cells were cotransfected by electroporation with either R1 and R3, or A1 and A3, and propagated in the presence of G418 (300 μg/ml) to create stably transfected cell lines. Stable transfection was verified by analyzing genomic DNA and mRNA (Figure S1). Since two different constructs were cotransfected for each condition, combinatorially it would be possible to generate three different retrocyclin peptides as illustrated in Figure 1B. For example, if cells were cotransfected with the R1 and R3 constructs, they could theoretically generate a heterodimer (HL60 cells containing retrocyclin constructs R1 and R3 [R1R3]) or homodimers (R1R1 or R3R3).

Figure 1. Design of Retrocyclin Constructs

(A) Shows a schematic of the four constructs (R1, R3, A1, and A3) used for stable transfections along with native retrocyclin cDNA. All constructs have two termination codons at the end to ensure read-fidelity (red). Constructs A1 and A3 contain additional downstream residues (orange), whereas constructs R1 and R3 lack them. The two arrows indicate the position at which the two site-directed mutagenesis (⊗17Q and R70K) were performed.

(B) Shows the three possible mature retrocyclin peptides that could be formed from the constructs, homodimers of R1 or A1 encoding RC-100 (wild-type retrocyclin), heterodimers of A1 and A3 or R1 and R3 encoding RC-101 (single lysine congener), and homodimers of R3 or A3 encoding RC-101_2K (double lysine congener).

Extracts of Promyelocytic Cells Stably Transfected with Retrocyclin Constructs Are Active against HIV-1

We next analyzed if correcting the termination codon in the retrocyclin constructs could restore the translation of biologically active retrocyclin peptides. The infection of TZM-bl cells with HIV-1 BaL was significantly reduced when cells were treated with cellular acid extracts of R1R3 cells (p < 0.004) and HL60 cells containing retrocyclin constructs A1 and A3 (A1A3) (p < 0.002) (Figure 2A). A standard tetrazolium MTT assay revealed that the extracts did not affect cellular metabolism at the concentrations used in the experiment (Figure 2E). Addition of A1A3 cell extracts to HIV-1 infected PM1 cells (Figure 2B) and PBMCs (Figure 2C) showed significant (p < 0.002 and p < 0.004, respectively) decrease in the viral titer as compared to cells treated with control HL60 cell extract. A trypan blue exclusion assay was performed in PBMCs to monitor cell viability (Figure 2F). We next affinity purified R1R3 and A1A3 cell extracts using anti-RC-101 antibody and confirmed the antiviral activity in a luciferase-based assay system (Figure 2D). Interestingly, A1A3 cell extracts were found to be consistently more active than equivalent amounts of R1R3 cell extract, which suggests a role for the downstream residues in retrocyclin processing. These results indicate that biologically active recombinant retrocyclin peptides can be synthesized in human promyelocytic cells. As a next step we tested the presence of retrocyclin in promyelocytic cells using immunostaining.

Figure 2. Extracts from HL60 Cells Stably Transfected with Retrocyclin Constructs Are Active against HIV-1 Infection

(A) TZM-bl cells were treated with extracts or peptide as indicated in the figure and infected with HIV-1 BaL (6.5 ng/ml p24) for 24 h. Infection was measured as percent luciferase activity compared to cells treated with control cell extract (average relative luciferase units [RLUs] of control HL60 extract = 178,200).

(B, C) PM1 cells and PBMCs were treated with extracts or peptide as indicated and infected with HIV-1 BaL (2 ng/ml p24) and cultured for 5–9 d. Bars represent percent BaL HIV-1 levels in the supernatants collected on days 5 (B) and 9 (C). The amount of p24 in PM1 cells treated with control extract = 76.85 ng/ml and in PBMCs treated with control extracts = 55.99 ng/ml.

(D) TZM-bl cells were treated with immunopurified (IP) extracts or peptides as indicated and infected with BaL HIV-1 (p24 = 2 ng/ml) for 24 h. Infection was quantified as percent luciferase activity compared to cells treated with control HL60 cell IP extracts (average RLU = 764,460). Error bars represent standard error of the mean (SEM). n = 3–4; #, p < 0.004; *, p < 0.002; **, p <0.0005.

(E) Cellular viability of TZM-bl cells treated with HL60 acid extracts as indicated was determined by measuring the reduction of MTT after 24h (n = 3). Bars represent percent viability as compared to vehicle control and error bars represent SEM.

(F) Cell death was monitored in PBMCs treated with the acid extracts by a trypan blue exclusion assay on day 9 (n = 1).

Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides

Immuno-dotblot analyses revealed that our anti-RC-101 antibody specifically recognized lysine-containing human retrocyclin analogs (synthetic RC-101 and RC-101_2K) and RC-100 (i.e., wild-type form) to a lesser extent (Figure 3A) but not human neutrophil peptides 1–3, or peptides with very similar tertiary structure including rhesus theta defensin-1 (RTD-1) and protegrin-1 (PG-1) (Figure 3B). This antibody was used to visualize the expressed retrocyclin peptides in the stably transfected HL60 cells by immunofluorescence staining, which revealed that R1R3 cells and A1A3 cells were brightly stained as compared to vector control (VC) cells (Figure 3C). Slides treated with preimmune serum showed no staining (unpublished data). Note that the staining of A1A3 was brighter than R1R3 and the morphology of A1A3 cells was smaller than VC cells. Experiments were next designed to purify and confirm the identity of the expressed retrocyclin peptides from the cell extracts.

Figure 3. Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides

(A) Retrocyclin peptides RC-100, RC-101, and RC-101_2K peptides (in duplicates) and (B) RC-100, RC-100b, RC-101, protegrin-1 (PG-1), rhesus theta defensin-1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3) were dotted (0–8 ng/4 μl dot) on a PVDF membrane and subjected to immuno-dotblot analysis.

(C) VC, R1R3, and A1A3 (100,000 cells each) were fixed onto glass slides and incubated with rabbit anti-RC-101 antibody followed by biotinylated goat anti-rabbit IgG secondary antibody and then stained using fluorescein isothiocyanate (FITC)-avidin. Slides were visualized using Zeiss Axiovert 200M microscope system at 40× magnification. The three rows show FITC staining, DIC, and the merged image, respectively. Scale bar represents 20 μm.

Stably Transfected Promyelocytic Cells Produce Retrocyclin Peptides

Reverse-phase high-performance liquid chromatography (RP-HPLC) was utilized to purify the recombinant retrocyclin peptides from stably transfected HL60 cell extracts. Figure 4A shows the RP-HPLC trace of A1A3 and synthetic RC-101. Synthetic RC-101 was recovered in fractions collected at 26–28 min. A1A3 HPLC Fractions collected from 23–30 min were analyzed on a 16% Tricine-SDS-gel. Control samples did not contain any protein bands at the expected size, whereas fractions from R1R3 cell extracts revealed protein bands of about 6-kDa size (unpublished data). Interestingly, A1A3 HPLC fractions revealed multiple protein bands, which we further analyzed by western blot (Figure 4B). The western blot analysis revealed bands at sizes corresponding to a monomer, dimer, and trimer of retrocyclin. Interestingly, the presence of multimeric forms of retrocyclin has been independently observed by Daly and colleagues [29]. Furthermore, the RP-HPLC purified A1A3 fractions inhibited entry of HIV-1 BaL in TZM-bl cells (Figure 4C). The IC₅₀ of retrocyclin peptides expressed by A1A3 cells (2 μg/ml) was similar to that of synthetic RC-101 (1.25 μg/ml) [8].

Figure 4. Stably Transfected Promyelocytic Cells Produce Retrocyclin

(A) Shows the RP-HPLC trace of A1A3 cell extract (from 10⁸ cells) and 50 μg of synthetic RC-101.

(B) Western blot of A1A3 HPLC fractions (23–28 min) using rabbit anti-RC-101 antibody. The arrows indicate the multimeric forms of retrocyclin observed in A1A3 fractions.

(C) TZM-bl cells were infected with HIV-1 (p24 = 2 ng/ml) in the presence or absence of pooled A1A3 fractions (final dilution 1:6 in D10) or 2 μg/ml of RC-101 for 24 h. Infection was quantified by luciferase measurement (average RLU of vehicle control with virus = 85,450).

Error bar represents SEM and n = 3–6; *, p < 0.0015; **, p <0.0001. MALDI-TOF MS spectra of Lys-C digested (D) synthetic RC-101_2K, (E) synthetic RC-101, and (F) A1A3 HPLC fraction 26 reveal that A1A3 cells produce RC-101.

To determine the identity of the retrocyclin peptide expressed by A1A3 cells, HPLC fraction 26 was analyzed by mass spectrometric analysis (MALDI-TOF-MS) at the Microchemical and Proteomics Facility, Emory University (Atlanta, Georgia, US). Analysis of A1A3 Fraction 26 revealed peaks with masses 1,889.775 Da (oxidized) and 1895.890 Da (reduced), which is nearly identical to the expected mass of synthetic cyclic RC-101 (1,889.85 Da and 1,895.96 Da, respectively; unpublished data) and is in agreement with reduction of the three disulfide bridges in the molecule. Furthermore, treatment with iodoacetamide yielded mass species of 2,238.081 Da for the A1A3 fraction 26 and 2,238.071 Da for RC-101 corresponding to the predicted 6-fold–alkylated form of RC-101 (expected mass = 2,238.097 Da). Comparison of spectrum of the Lys-C digest of reduced/alkylated synthetic RC-101_2K (peak at 1,123.577 Da; peptide cleaved at two Lys-Gly bonds; Figure 4D), synthetic RC-101 (peak at 2,256.097 Da; peptide cleaved at a single Lys-Gly bond; N-terminal sequence determined as: Gly-Ile-Cys-Arg-; Figure 4E), and A1A3 fraction 26 (peak at 2,256.010 Da) suggests that the A1A3 cells are expressing RC-101 (Figure 4F). These data confirmed that correctly folded mature retrocyclin peptides can be expressed by human cells. In the following experiments we explored alternative methods to express the peptide endogenously. Of particular interest was the effect of aminoglycosides in mediating varying degrees of termination codon read-through as previously described [19–25].

Aminoglycosides Mediate Read-Through of Termination Codon of Retrocyclin Gene and Restore Anti-HIV-1 Activity

We tested the ability of three commonly used aminoglycosides (gentamicin, amikacin, and tobramycin) to induce termination codon read-through of retrocyclin cDNA. The native retrocyclin gene was fused with a luciferase reporter at the C terminus to create two constructs: unrescued RC-101 and rescued RC-101 (positive control) as shown in Figure 5A. These constructs were transfected into HOS-CD4-CCR5 cells, grown in the presence of varying concentrations of aminoglycosides, and the degree of read-through quantified by measuring luciferase. Application of tobramycin (10 μg/ml) was the most effective, producing a 26-fold increase in read-through (p < 0.0007; Figure 5B).

Figure 5. Aminoglycosides Mediate Read-Through of the Premature Termination Codon within the Retrocyclin Gene and Promote Anti-HIV-1 Activity

(A) Shows a schematic of the luciferase fusion constructs unrescued RC-101 and rescued RC-101 along with native retrocyclin cDNA.

(B) HOS-CD4-CCR5 cells cultured in antibiotic free medium (D10⁻) were transfected with unrescued RC-101 (negative control) or rescued RC-101 (positive control) plasmids along with phRL-CMV vector (transfection control). The next day transfected cells were treated with PBS for control cells or aminoglycosides at the indicated concentrations and allowed to grow for 24 h. Read-through was determined by measuring luciferase levels. Data are expressed as fold increase in luciferase expression normalized to renilla levels.

(C) TZM-bl cells grown in D10⁻ were treated for 30 min with PBS, RC-101 (2.5 μg/ml), RC-100 (2.5 μg/ml), or aminoglycosides as shown in the figure and infected with HIV-1 BaL (2 ng/ml p24) for 24 h followed by luciferase measurement. Error bars represent SEM. n = 3–6; #, p < 0.007; * p < 0.0005; ** p <0.0001.

(D) TZM-bl cells cultured on cover slips were treated with PBS (Con) or 10 μg/ml tobramycin (Tob) and then immunostained with rabbit preimmune or antiretrocyclin serum using a biotinylated secondary antibody FITC-avidin system.

(E) Cellular cytotoxicity was assessed by performing an MTT assay on TZM-bl cells treated with indicated amount of peptide or aminoglycosides (n = 3). Bars represent percent metabolic inhibition as compared to control (vehicle + virus).

(F) TZM-bl cells, treated with either PBS, tobramycin (10 μg/ml), or RC-100 (2.5 μg/ml), were incubated with preimmune serum or antiretrocyclin serum as indicated and infected with HIV-1 (p24 of 5 ng/ml). Data are represented as percent infection. Error bars represent SEM. n = 3; +, p < 0.018 Statistical significance was determined by two-tailed Student's t-test.

Having thus established the optimal aminoglycoside concentration required to achieve read-through of retrocylin cDNA, we next determined if aminoglycosides could restore the translation and anti-HIV-1 activity of native retrocyclin peptides. HeLa-derived cells lines such as TZM-bl cells can natively express retrocyclin mRNA (unpublished data). We applied aminoglycosides to TZM-bl cells and challenged them with HIV-1 BaL. We found that cells treated with gentamicin and tobramycin significantly (p < 0.0005 and p < 0.0001, respectively) inhibited HIV-1 infection as compared to untreated cells (Figure 5C). The effect was modest when compared to inhibition by synthetic peptides. Cell viability, determined by a tetrazolium-based MTT assay, was not affected by the application of aminoglycosides at the mentioned concentrations (Figure 5E).

In order to visualize the retrocyclins expressed by application of aminoglycosides, we performed immunostaining. TZM-bl cells were treated with PBS control or 10 μg/ml tobramycin and stained with anti-retrocyclin antibody or preimmune serum. Control cells showed no staining while cells treated with tobramycin revealed brightly stained cells suggesting that aminoglycosides can induce the expression of retrocyclin peptides (Figure 5D).

We next incubated TZM-bl cells with tobramycin (10 μg/ml) for 24 h, and then treated the cells with preimmune or anti-retrocyclin serum followed by infection with HIV-1. Figure 5F reveals that cells treated with preimmune serum showed a modest yet significant reduction in infection as compared to cells treated with anti-retrocyclin antibodies (p < 0.018), suggesting that the antibody inhibited the endogenous retrocyclins. These data confirm that the anti-HIV-1 activity observed is due to the endogenous retrocyclin peptides expressed when tobramycin was applied to cells.

Aminoglycosides Induce Production of Retrocyclin Peptides in Cervicovaginal Tissues

We next analyzed the ability of aminoglycosides to induce the expression of retrocyclin peptides in an organotypic model cervicovaginal tissue. Tissues were treated apically with tobramycin or control (PBS) for 24 h and anti-retrocyclin immunohistochemical analysis was performed. Interestingly, tissues treated with tobramycin alone and stained with anti-retrocyclin antibody revealed brightly stained cells (Figure 6A) suggesting that production of retrocyclin peptides is induced upon application of aminoglycosides. Lactate dehydrogenase (LDH) activity in the medium underlying the tissues was performed to determine tissue cytotoxicity. The LDH assay revealed that application of 10 μg/ml tobramycin was not cytotoxic to the tissues (Figure 6B). In addition, treatment of tobramycin did not affect the metabolic activity adversely, which was determined by an MTT assay performed on one tissue (unpublished data).

Figure 6. Expression of Retrocyclins in Cervicovaginal Tissue Model Using Aminoglycosides

(A) Cervicovaginal tissues were treated with PBS (Con) or 10 μl tobramycin (Tob) and incubated with rabbit preimmune serum or antiretrocyclin antibody. The slides were then incubated with biotinylated goat anti-rabbit IgG secondary antibody and then stained using FITC-avidin.

(B) Cytotoxicity was determined by measuring LDH activity in media underlying the tissues treated with PBS or tobramycin as indicated. Bars represent absorbance measured as 490 nm and error bars represent SEM; n = 6.

(C) HPLC trace of extracts of tissues treated with 10 μg/ml tobramycin (tissue + Tob) and 20 μg of synthetic RC-100.

(D) RC-100 synthetic peptide (indicated amounts), HPLC fractions 27–29 of control, tobramycin-treated, and RC-100 were dotted on a PVDF membrane and analyzed by immuno-dotblot.

In order to purify endogenous retrocyclins expressed in the tissues, we utilized RP-HPLC. Figure 6C shows an HPLC trace of control, tobramycin-treated tissue extracts as compared to synthetic RC-100 peptide. Synthetic RC-100 peptide was recovered in fractions collected at 27–29 min. Corresponding fractions from control and tobramycin-treated tissues were analyzed by immuno-dotblot analysis using the anti-RC-101 antibody. Figure 6D shows that retrocyclin peptides were recovered in fractions 27–29 min in tobramycin-treated tissue samples but not in control tissue samples. The amount of retrocyclin (RC-100) expressed in tobramycin-treated cervicovaginal tissues was estimated by densitometry to be approximately 1.6 μg/tissue. Together these studies show that aminoglycosides are promising molecules to suppress the premature termination codon of retrocyclin transcripts and restore the ability of cervicovaginal tissues to protect cells from HIV-1.

Conclusion

Identifying effective drugs to prevent HIV-1 infection and other viral infections is essential for countering the spread of these diseases. Exogenous (synthetic) retrocyclins exhibit full activity in the complex environment of vaginal fluid and the peptide is very well tolerated in organotypic human cervicovaginal tissue models [17]. Moreover, HIV-1 evolves little resistance during continued passaging in the presence of the peptide [18]. For these and other reasons, retrocyclins have emerged as potential topical microbicides to protect against sexually transmitted HIV-1 infections.

In this study we have taken a different path towards developing θ-defensin therapeutics. The human pseudogenes that encode the demidefensin precursors whose post-translational processing gives rise to mature retrocyclin are expressed at the mRNA level in multiple organs, including the spleen, bone marrow, thymus, testis, and skeletal muscle [14], and cervicovaginal epithelia (A. M. Cole, unpublished data). By transfecting human myeloid cells with plasmids containing retrocyclin genes without a premature termination codon, we demonstrated that the “machinery” needed to process, trim, splice, and oxidize retrocyclin precursors was available in human myeloid cells. Two sets of expression constructs were transfected into cells: a shorter form (R1R3) that terminates at the end of the retrocyclin gene and a longer form that contains (A1A3) additional 3′ untranslated residues (UTR). Interestingly, A1A3 cells expressed higher levels of retrocyclin peptides as compared to R1R3 cells indicating a role for additional residues in the translational efficiency of these peptides. This was not altogether surprising as other studies have shown that the length of the 3′-UTR regulates translation efficiency [28,30]. Finally, we showed that aminoglycoside-treated cells and cervicovaginal tissues could produce retrocyclins endogenously by suppressing the premature termination codon in their endogenous mRNA transcript.

Since approximately 30% of inherited disorders may result from premature termination codon mutations, there has been tremendous interest and some progress in developing and applying agents that can read-through premature UAA, UAG, or UGA termination codons [25]. Although aminoglycosides, as used in this study, have been most widely investigated, exciting new agents such as PTC-124, have also appeared [31,32]. In a sense, human retrocyclin-deficiency is also an inherited disorder, albeit one with an incidence of 100%. It is caused by a premature termination codon mutation that occurred after human lineage diverged from the lineage we share with orangutans, lesser apes, and old world monkeys. Since HIV-1 and other viruses that currently infect humans have evolved in the absence of selective pressure exerted by retrocyclins, the ability to reawaken this ancestral molecule could be used to strengthen the innate immune system's ability to prevent or limit the infections they now induce.

Materials and Methods

Maintenance of cells, tissues, and viruses.

HL60 cells [33,34] obtained from ATCC were cultured in Iscoves's DMEM with 20% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (I20). TZM-bl cells [35] stably expressing CD4, CCR5, and CXCR4, has firefly luciferase gene under the control of HIV-1 promoter (from J. C. Kappes, X. Wu, and Tranzyme Inc). TZM-bl, HOS-CD4-CCR5 [36,37] (from N. R. Landau), PM1 cells [38], (from M. Reitz), and HIV-1 BaL, an R5 tropic strain, were all procured through the National Institutes of Health (NIH) AIDS Research and Reference Reagent program. HIV-1 BaL viral stocks were prepared by infecting PM1 cells [18]. PBMCs were isolated from blood drawn from a healthy HIV-1 seronegative donor as per the guidelines of the institutional review board of University of Central Florida. PBMCs were isolated using Lymphosep (MP biomedicals LLC), and cultured in RPMI-1640 medium with 10% FBS (R10) supplemented with 50 units of IL-2 (R10-50U) and 5 μg/ml of phytohemagglutinin (PHA) for 3 d. The cells were then resuspended in R10-50U at a density of 0.8 × 10⁶ cells/ml and grown for 5–6 d.

Cervicovaginal tissues (EpiVaginal) were obtained from MatTek Corporation and maintained in proprietary growth medium as per the company's guidelines. The tissues were composed of a full-thickness, stratified vaginal-ectocervical layer intermixed with Langehans cells and underlying lamina propria. The tissues were allowed to grow on transwell cell culture inserts at the air-liquid interface.

Creation of retrocyclin constructs and stably transfected HL60 cells.

Retrocyclin cDNA was amplified from human bone marrow cDNA and cloned into pCRII-TOPO vector (Invitrogen). Two mutations, Termination codon (⊗17) → Gln (Q17) and Arg (R70) → Lys (K70) were introduced, either ⊗17Q alone (RC-100) or both (RC-101) using Quick change site-directed mutagenesis (Stratagene) and subcloned in-frame into the phCMV-luc-FSR vector (Genlantis) to generate four constructs R1, R3, A1, and A3 (Figure 1A). Plasmids R1 and A1 encode RC-100 nonapeptide while R3 and A3 encode RC-101 nonapeptide. Constructs A1 and A3 have a longer insert that includes additional downstream residues. HL60 cells (10⁷ cells/400 μl Iscove's DMEM) were cotransfected with 2 μg each of linearized R1, R3 or A1, A3, or phCMV-luc vector alone, by electroporation (exponential decay wave mode- 280 V; 975 μF) and selected in I20 medium with 300 μg/ml G418 sulfate. Stable transfectants thus produced were named according to the constructs used for cotransfection (R1R3, A1A3, or VC). Presence of these constructs in the cells was verified using PCR of genomic DNA (Figure S1A). PCR conditions used were the following: initial denaturation at 95 °C for 3 min; 30 cycles of 95 °C for 1 min; 58 °C for 1 min; 72 °C for 2 min; followed by a final extension at 72 °C for 7 min. Sequences of the primers used for the PCR reaction are listed in Table S1. RNA was extracted from 10⁶ cells (HL60, VC, R1R3, and A1A3) using TRIzol (Invitrogen), cleaned with DNaseI (Ambion Inc.), and cDNA synthesized (iScript, BioRad). Expression of recombinant genes was verified by PCR from the cDNA and subsequent restriction digestion using HpyCH4V (New England Biolabs) (Figure S1B and S1C).

Acid extraction and affinity purification of retrocyclin peptides.

HL60 cells (control, VC, R1R3, and A1A3) were extracted with 5% acetic acid by vortexing for 20 min, centrifuged for 10 min at 10,000g, supernatants were then vacuum-dried and resuspended in 0.01% acetic acid. HL60 acid extracts (equivalent of 20 × 10⁶ cells) were affinity purified using anti-RC-101 polyclonal antisera immobilized to a Carbolink coupling gel (Pierce Biotechnology Inc.) prepared according to the manufacturer's instructions. Immunopurified samples were desalted using Sep-Pak C-18 cartridges (Waters). The elutes were then dried and resuspended in 100 μl of 0.01% acetic acid. 100 μg of synthetic RC-101 peptide was also affinity purified as positive control (RC-101 IP).

Luciferase-based infection assay to determine anti-HIV-1 activity.

TZM-bl cells (4,000 cells/well; 96-well plate) were infected with HIV-1 BaL (2–6.5 ng/ml of p24^gag) in the presence of vehicle (0.01% acetic acid), HL60 extracts (from 0.25 × 10⁶ control, A1A3, or R1R3 cells), affinity purified extracts (from 0.625 × 10⁶ control HL60, VC, R1R3, A1A3 cells, or RC-101 IP diluted 1:32 times), or RC-101 (20 μg/ml) (positive control) for 24 h. Treatments were then removed and the infection was quantified by measuring luciferase using Bright-Glo reagents (Promega) in an LMax luminometer (Molecular Devices). Cytotoxicity and metabolic activity of cells were verified by a tetrazolium-based MTT assay (R&D Systems) performed on identically treated cells.

Antiviral infection assay in suspension cells and HIV-1 p24^gag ELISA.

Acid extracts of stably transfected HL60 cells were vacuum-dried and resuspended in PBS. PM1 cells (10⁵ cells) or PBMCs (10⁶ cells) were treated with PBS (vehicle) or HL60 extracts (from 10⁴ cells for PM1 and 10⁵ for PBMCs) of control cells or A1A3 cells or 10 μg/ml of synthetic RC-101 and infected with HIV-1 BaL (2 ng of p24/ml) in 100 μl of RPMI medium with 20% FBS (R20) for 2 h. Cells were then washed with 2 ml of R20, resuspended in fresh medium containing the treatments, and cultured for 5–9 d. Subsequently on alternate days culture supernatants were collected and fresh medium with the corresponding treatments was added. Viability of the cells was measured using trypan blue exclusion assay. Amount of HIV-1 virus in the culture supernatants was quantified by ELISA for HIV-1 p24^gag (Perkin Elmer).

Immuno-dotblot assay.

Peptides RC-100, RC-100b, RC-101, RC-101_2K, synthetic protegrin-1 (PG-1), Rhesus theta defensin–1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3), or unknown samples were dotted (4 μl dot) as indicated on a 0.22-μm polyvinylidene fluoride (PVDF) membrane (Immobilon-P) that was activated with methanol and presoaked in TBS. The membrane was then probed with 1:1,000 rabbit anti-RC-101 antibody and developed using Immun-star HRP reagent (BioRad) [17].

Immunostaining of stably transfected HL60 cells using anti-RC-101 antibody.

HL60 cells (VC, R1R3, and A1A3) were fixed on slides (100,000 cells/slide), immersed in 10% Formalin in PBS for 10 min, washed (PBS for 2 min), incubated in Target retrieval solution (Dako North America Inc.) for 20 min at 95 °C, cooled to 25 °C, washed, blocked (2% Goat Serum, 0.1% Triton-X, 0.05% Tween-20, antibody buffer [10 mg/ml BSA/1 mg/ml gelatin/PBS]) for 20 min, and incubated in rabbit preimmune serum or rabbit anti-RC-101 antibody (1:5,000 in antibody buffer) overnight. Slides were washed, incubated in biotinylated goat anti-rabbit IgG antibody (1:20,000 in 1% goat serum/PBS for 30 min), followed by additional washing and treatment with Fluorescein-Avidin D (Vector Laboratories Inc.; 1:500 in PBS for 30 min). Cover slips were mounted using Vectashield fluorescence mounting medium and visualized using a Zeiss Axiovert 200M microscope system.

Tissues for immunofluorescence staining were fixed in 4% paraformaldehyde and slides were prepared by Mass Histology. The slides were deparaffinized, washed with TBS, and stained with anti-retrocyclin or preimmune serum and immunostained the same way as cells. The slides were then visualized on a Zeiss Axiovert 200M microscope system with 450 ms exposure time for all slides.

Separation of proteins from stably transfected HL60 extracts using RP-HPLC.

Acid extracts from control HL60 and A1A3 cells (equivalent of 100 × 10⁶ cells) were separated by RP-HPLC using the Alliance HT Waters 2795 Separations Module on a C₁₈ Column equilibrated in solvent A (aqueous 0.1% TFA). Elution was done with a gradient of 0%–95% solvent B (0.08% TFA in acetonitrile), for 75 min, at 1 ml/min. Collected fractions (1 ml each) were vacuum dried and reconstituted in 100 μl of 0.01% acetic acid. Synthetic RC-101 peptide (control) was recovered from the fractions eluting at 26–28 min. A1A3 HPLC fractions (numbers 23–28) were electrophoresed on a 16% Tricine-SDS gel and electroblotted on a 0.22 μm PVDF membrane at 180 mÅ for 22 min. The western blot membrane was then processed as described [17] and developed with ChemiGlow reagent (Alpha Innotech). A1A3 RP-HPLC fractions (27–30 min) were pooled and the concentration was determined to be (2.13 ng/μl) by densitometry measurements using Quantity one 1-D analysis (BioRad). A luciferase-based assay was used to verify the activity of A1A3 HPLC fractions (diluted three times in D10) against HIV-1 BaL (2 ng p24/ml).

MatTek cervicovaginal tissues treated with PBS (control) or 10 μg/ml tobramycin were extracted with T-PER reagent (Pierce Biotechnology Inc.) and separated by RP-HPLC. 20 μg of synthetic retrocyclin (RC-100) was also separated as a positive control. Synthetic RC-100 was eluted in fractions collected at 27–29 min. Tissue samples eluted at 27–29 min were vacuum-dried to near dryness and resuspended in 100 μl of 0.01% acetic acid. HPLC fractions (27–29 min) of MatTek tissue extracts (control or tobramycin-treated) and synthetic RC-100 were analyzed by immuno-dotblot analysis.

Mass spectrometric analysis.

A1A3 HPLC Fraction 26, RC-101, and RC-101_2K were reduced, alkylated, and treated with Lys-C protease for 30 min before analyzing by mass spectrometry. In brief, 20 mM Tris [2-carboxyethyl] phosphine (TCEP) was used to reduce (30 min at 25 °C) the samples, alkylated by incubating the samples with iodoacetamide (60 mM; 45 min at 25 °C [pH 8–9]) followed by digestion with Lys-C (Wako Chemicals; 30 min at 37 °C) and subjected to MALDI-TOF-MS analysis using a model 4700 Proteomics Analyzer (Applied Biosystems) as described previously [39]. Lys-C digested RC-101 was desalted using C18 ZipTip (Millipore Corp.) and subjected to Edman degradation on cLC-Procise sequencer (Applied Biosystems).

Aminoglycoside mediated read-through of termination codon.

Wild-type and mutant retrocyclin cDNAs were subcloned into phCMV-luc-FSR vector to create unrescued RC-101 and rescued RC-101 C-terminal luciferase fusion constructs, and verified by sequencing. HOS-CD4-CCR5 cells were cultured in antibiotic free growth medium (D10⁻) and cotransfected with 0.5 μg of unrescued or rescued (positive control) RC-101 plasmids along with 0.1 μg of phRL-CMV vector (transfection control containing renilla luciferase gene) using Effectene transfection reagent (Qiagen). The next day cells were treated for 24 h with the appropriate aminoglycoside (40 μg/ml amikacin or 5 μg/ml gentamicin or 10 μg/ml tobramycin) or D10⁻ for control cells. Read-through was determined by measuring luciferase and renilla levels using a dual luciferase assay (Promega).

TZM-bl cells (4,000 cells/well; 96-well plate) were cultured in D10⁻ and treated with vehicle (PBS buffered D10⁻) or peptides RC-101 or RC-100 (2.5 μg/ml each) as positive control or aminoglycosides as before for 30 min followed by infection with HIV-1 BaL (p24 of 2 ng/ml) at 37 °C for 24 h. Subsequently, viral infection was quantified by measuring luciferase levels using Bright-Glo reagents (Promega). Cellular metabolism was monitored by measuring reduction the ability of cellular dehydrogenases to reduce MTT to formazan (R&D Systems).

TZM-bl cells were cultured on coverslips and treated with PBS control or 10 μg/ml of tobramycin for 24 h. The coverslips were then processed for immunofluorescence staining with anti-retrocyclin (rabbit anti-RC-101 antibody) or preimmune serum as described above.

For antibody-mediated neutralization experiments, TZM-bl cells (4,000 cells/well; 96-well plate) were cultured in D10⁻ medium and treated with vehicle (PBS) or 10 μg/ml of tobramycin for 24 h. The next day, cells were treated with either rabbit preimmune or anti-retrocyclin serum diluted 1:10 in D10⁻ medium containing tobramycin or RC-100 (2.5 μg/ml). 2 h later the cells were infected with HIV-1 BaL (p24 of 5 ng/ml) at 37 °C for 24 h. Viral infection was quantified as described above. An MTT assay was performed to confirm that the treatments were not cytotoxic (unpublished data).

Application of aminoglycosides to organotypic cervicovaginal tissue model.

Cervicovaginal tissues were treated topically with 100 μl of PBS (control; n = 4) or with 10 μg/ml of tobramycin (n = 8) for 24h. Viability was assessed on control and tobramycin-treated tissue (n = 1) using MTT assay kit (MatTek Corp.). Cytotoxicity was measured by quantifying LDH activity in the underlying medium collected 24 h after treatment with PBS or tobramycin by using CytoTox96 nonradioactive cytotoxicity assay kit (Promega Corp.).

Supporting Information

Figure S1. Verification of Stable Transfection of Retrocyclin Constructs

Analysis of the genomic DNA and RNA of transfected HL60 cells confirms the stable transfection and transcription of rescued retrocyclin constructs, respectively. (A) PCR on genomic DNA template from transfected HL60 cells shows a 215-bp fragment representing retrocyclin cDNA construct and a 890-bp fragment of native retrocyclin gene in the genomic DNA of A1A3 clones but not in the VC cells.

(B, C) Correction of the premature termination codon of retrocyclin cDNA introduces an additional HpyCH4V restriction site in the middle of an 87-bp cDNA fragment. RNA isolated from HL60 cells (control, R1R3 clones 1 and 2, and A1A3 clones 1 and 2) was used to make cDNA. Retrocyclin constructs were amplified by PCR using the cDNA as template and digested using HpyCH4V restriction enzyme. Electrophoresis of the digested PCR products shows the expected 87-bp fragment in control cells (B) and the expected absence of 87-bp fragment in R1R3 and A1A3 clones (C). All the products were also verified by DNA sequencing.

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Table S1. Primers Used for Verification of Retrocyclin Constructs

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Acknowledgments

The authors thank Karthikeyan Sivaraman for helpful discussions and Gerry Quinn for assistance with RP-HPLC purification. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Author contributions. ALC and AMC conceived and designed the experiments. NV, ALC, PR, RIH, and OS performed the experiments. NV, ALC, PR, AJW, RIH, JP, and AMC analyzed the data. PR, AJW, RIH, OS, and JP contributed reagents/materials/analysis tools. NV and AMC wrote the paper.

Stroke Vaccine Shows Promise

JN International Medical Corporation in Omaha, Nebraska has developed a vaccine against Stroke. While the repeated animal studies suggest the vaccine works in pigs and their anatomy is closer to human. Strokes begin when inflammation occurs on a molecular level in blood vessels as part of a response by the immune system. During a 5 year long study, the pigs that received treatment with the vaccine had fewer strokes caused by blood clots than those that received Placebo. None of the pigs that had treatment suffered from a hemorrhagic stroke, in which blood vessels rupture and memory loss. Much more study will be needed before the vaccine can be tested in humans. During suggests that the first patients to test should be those with an exceptionally high risk of brain damage from a stroke because they have inoperable brain aneurysms -- small blood vessels that balloon out and threaten to burst. Full scale Phase-1 study is proposed to be conducted in 2009-2010 in the United States. The vaccine technology is filed for patent with United States Patent Office and in 136 PCT countries. The stroke vaccine development technology can be read from web page of World Intellectual Property Organization (WIPO) http://www.wipo.int/pctdb/en/wo.jsp?WO=2008121146

Omaha, Nebraska -- JN's Stroke vaccine could offer a "new dimension" in stroke prevention. The vaccine would most likely be targeted to people who are susceptible to strokes. Epilepsy, stroke, Parkinson's disease, Huntington, chronic pain, depression -- all might one day be treated and/or prevented with a new vaccine said Dr. Jeeri R. Reddy, the President and Scientific Director of the corporation.

Impact of Stroke:
Strokes occur in one of two ways; a blood clot forms and then cuts off circulation to part of the brain or a blood vessel ruptures and leaks blood into the brain. In both cases, a stroke can lead to permanent disability or death.

Stroke is the third leading killer in the United States, resulting in the following statistics annually: •750,000+ victims;

---    160,000 deaths (3rd leading cause of death);

---    266,000 survivors with permanent disabilities;

---    30,000 new permanent admissions to nursing homes;

---    Over 4 million living survivors of stroke;

---    Every 45 seconds in the USA, someone has a stroke;

---    Huge economic impact with costs of $40-$70 billion per year.

"Vaccines have always been an integral part of JN's business and our contribution to public health and saving lives and the startup of this new facility in Omaha intends to provide quality vaccines that prevent diseases and enhance health care for millions around the world. Vaccination prevents millions of deaths per year, and yet millions more are dying due to the lack of vaccines. Stringent regulatory hurdles and the perception of moderate returns on investment have not encouraged companies like us to enter the markets faster. Prevention is better than cure --innovation of new vaccines for healthcare make a real difference for the world. To realize this goal, we recognize that JN resources and capabilities are necessary, but not sufficient--we just can't do it alone. Successful, productive partnerships are essential for JN's ongoing pursuit of innovation and excellence," said Dr. Reddy.

JN-International Medical Corporation:

JN-International Medical Corporation is a U.S. based biopharmaceutical corporation which since 1998 has been focused on developing vaccines and diagnostics for infectious disease for developing countries. An Inc 5000 corporation, making it the 95th ranking company in the United States and the 3rd biopharmaceutical global corporation, it produces Meningitis vaccine for which it holds the international intellectual property rights (IPR).

JN boasts a decade of achievements including prevention of mother-to-child transmission of HIV in South East Asia, development of inexpensive vaccine for Meningococcal meningitis serogroups A, C, Y&W-135 and lately the discovery of a new generation preventive and therapeutic vaccine for Stroke and neurological disorders. The R&D division of JN-International is currently researching and developing vaccines for the prevention and cure of Hepatitis C, Herpes, Tuberculosis and a preventive and therapeutic vaccine for major Neurological disorders. JN-International Medical Corporation (JNI), based in Omaha, Nebraska, USA.

Big Head, Bald Head - Is Hair Loss Caused by Skull Expansion?

A new study suggests that people who agonize over hair loss (known as androgenetic alopecia) might have their big heads to blame. A UK researcher has determined that men and women whose skull bones keep growing during adulthood are far more likely to develop male and female pattern baldness.

The Medical Hypotheses journal reports a possible breakthrough in our understanding of androgenetic alopecia which could one day lead to an alopecia cure.

Paul Taylor from Top Hair Loss Remedy.com spent seven years of independent research into androgenetic alopecia following concerns about his own hair loss. In his article, he explains how both male and female thinning hair and baldness involves the continued growth of the skull bones that lie directly under the scalp where hair loss develops. This bone growth is called skull expansion.

Skull expansion seems to have finally solved certain mysteries that have been baffling hair loss boffins for years. These include why the same shape (or pattern) of hair loss will always develop in men with extreme baldness. Severe hair loss at the front (frontal baldness) and crown of the head will leave just a small horseshoe shaped area of hair growing at the back and sides. Skull expansion explains why this happens - something the current theory for hair loss cannot do.

Another mystery involves the male sex hormone dihydrotestosterone (DHT). Male pattern baldness has been linked to DHT, but this hormone also causes body and facial hair to grow at puberty. Skull expansion explains how DHT is connected to both hair loss and hair growth.

If this theory for androgenetic alopecia by skull expansion is confirmed, it could lead to a total redirection of genetic research, affect future treatment methods and might eventually lead to an alopecia cure.

Disease diagnosis in just 15 minutes

Disease diagnosis in just 15 minutes

Testing for diseases such as cancer and multiple sclerosis could soon be as simple as using a pregnancy testing kit.

A team led by scientists at the University of Leeds has developed a biosensor technology that uses antibodies to detect biomarkers - molecules in the human body which are often a marker for disease – much faster than current testing methods.

The technology could be used in doctors’ surgeries for more accurate referral to consultants, and in hospitals for rapid diagnosis. Tests have shown that the biosensors can detect a wide range of analytes (substances being measured), including biomarkers present in prostate and ovarian cancer, stroke, multiple sclerosis, heart disease and fungal infections. The team also believes that the biosensors are versatile enough to test for diseases such as tuberculosis and HIV.

The technology was developed through a European collaboration of researchers and commercial partners in a 2.7 million Euro project called ELISHA.

ELISHA was co-ordinated by Dr Paul Millner from the Faculty of Biological Sciences at the University of Leeds, and managed by colleague Dr Tim Gibson. Says Dr Millner: “We believe this to be the next generation diagnostic testing. We can now detect almost any analyte faster, cheaper and more easily than the current accepted testing methodology.“

Currently blood and urine are tested for disease markers using a method called ELISA (Enzyme Linked Immunosorbant Assay). Developed in the 1970s, the process takes an average of two hours to complete, is costly and can only be performed by highly trained staff.

The Leeds team are confident their new technology – which provides results in 15 minutes or less - could be developed into a small device the size of a mobile phone into which different sensor chips could be inserted, depending on the disease being tested for.

“We’ve designed simple instrumentation to make the biosensors easy to use and understand,” says Dr Millner. “They’ll work in a format similar to the glucose biosensor testing kits that diabetics currently use.”

Professor Séamus Higson, Dean of the Faculty of Medicine and Biosciences, Cranfield Health, and one of the partners within the ELISHA programme, says: “The speed of response this technology offers will be of great benefit to early diagnosis and treatment of many diseases, and will permit testing in de-localised environments such as GP’s surgeries.”

A spinout company – ELISHA Systems Ltd – has been set up by Dr Gibson, commercial partners Uniscan Instruments Ltd and Technology Translators Ltd to bring the technology to market.

Says Dr Gibson: “The analytes used in our research only scratch the surface of the potential applications. We’ve also shown that it can be used in environmental applications, for example to test for herbicides or pesticides in water and antibiotics in milk.”

Neurosciences Institute Discovers Drug Extends Stroke Treatment Time to 24 Hours, Repairs Brain Tissue

Neurosciences Institute Discovers Drug Extends Stroke Treatment Time to 24 Hours, Repairs Brain Tissue

Scientists at the Blanchette Rockefeller Neurosciences Institute (BRNI) have discovered that Bryostatin - and a related class of drugs discovered at BRNI - administered 24 hours after stroke can rescue and repair brain tissue. These findings are markedly advanced compared to current stroke treatments that must be administered within three hours and are unable to repair damaged brain tissue.

Bryostatin could be an answer.

Morgantown, WV (PRWEB) September 3, 2008 -- Scientists at the Blanchette Rockefeller Neurosciences Institute (BRNI) have discovered that Bryostatin - and a related class of drugs discovered at BRNI - administered 24 hours after stroke can rescue and repair brain tissue. These findings are markedly advanced compared to current stroke treatments that must be administered within three hours and are unable to repair damaged brain tissue.

In an article to be published in the September 3 issue of the Proceedings of the National Academy of Sciences (PNAS), BRNI Scientific Director and Toyota Chair Daniel Alkon, M.D. and Professor Miao-Kun Sun, PhD describe how this Alzheimer's candidate drug repairs the brain and improves memory.

"Today's stroke patient has precious minutes to receive care without suffering irreversible damage or death. One of the greatest challenges in modern medical practice is finding an effective treatment that extends that treatment time and repairs damage," said BRNI Scientific Director Daniel Alkon, M.D. "Bryostatin could be an answer."

Bryostatin could be life changing for millions of Americans who suffer neurological conditions - from Alzheimer's disease to stroke. Each year in the United States, there are more than 780,000 strokes. It is the third leading cause of death in the country and the most common cause of long-term disability in developed countries. Nearly three-quarters of all strokes occur in people over the age of 65 and the risk of having a stroke more than doubles each decade after the age of 55.

"We're facing an aging Babyboomer population, an influx of 55-plus Americans, and treatment that is less time-restrictive and able to repair the brain if a stroke destroys tissue is urgently needed," said Alkon.

How Bryostatin Works:
Stroke symptoms typically develop rapidly - within seconds to minutes - and as oxygen becomes depleted in the brain tissue, cells die. This means that hundreds of thousands of neurons - each linked to thousands of connections - die. In animal testing, Bryostatin completely rescues these dying neurons, stimulates the growth of new connections and restores memory capacity. Additionally, the drug can be administered up to 24 hours following a stroke, increasing the number of patients it could potentially save.

This drug, suggests Alkon, offers potential to prevent and/or reverse brain degeneration not just in stroke victims, but also Alzheimer's disease and traumatic brain injury.

Previous studies had also shown Bryostatin's ability to accelerate the generation of new connections in the brain when paired with learning exercises. According to Alkon, this could eventually lead to new treatment therapies for children with compromised memory activity. BRNI is in discussion with the Food and Drug Administration (FDA) to begin clinical trials of the drug.

BLACK RASPBERRIES SLOW CANCER BY ALTERING HUNDREDS OF GENES

BLACK RASPBERRIES SLOW CANCER BY ALTERING HUNDREDS OF GENES

COLUMBUS, Ohio – New research strongly suggests that a mix of preventative agents, such as those found in concentrated black raspberries, may more effectively inhibit cancer development than single agents aimed at shutting down a particular gene.

Researchers at the Ohio State University Comprehensive Cancer Center examined the effect of freeze-dried black raspberries on genes altered by a chemical carcinogen in an animal model of esophageal cancer.

Garry D. Stoner

The carcinogen affected the activity of some 2,200 genes in the animals’ esophagus in only one week, but 460 of those genes were restored to normal activity in animals that consumed freeze-dried black raspberry powder as part of their diet during the exposure.

These findings, published in recent issue of the journal Cancer Research, also helped identify 53 genes that may play a fundamental role in early cancer development and may therefore be important targets for chemoprevention agents.

“We have clearly shown that berries, which contain a variety of anticancer compounds, have a genome-wide effect on the expression of genes involved in cancer development,” says principal investigator Gary D. Stoner, a professor of pathology, human nutrition and medicine who studies dietary agents for the prevention of esophageal cancer.

“This suggests to us that a mixture of preventative agents, which berries provide, may more effectively prevent cancer than a single agent that targets only one or a few genes.”

Stoner notes that black raspberries have vitamins, minerals, phenols and phytosterols, many of which individually are known to prevent cancer in animals.

“Freeze drying the berries concentrates these elements about ten times, giving us a power pack of chemoprevention agents that can influence the different signaling pathways that are deregulated in cancer,” he says.

“What’s emerging from studies in cancer chemoprevention is that using single compounds alone is not enough. And berries are not enough. We never get 100 percent tumor inhibition with berries, so we need to think about another food that we can add."

---

To conduct this study, Stoner and his colleagues fed rats either a normal diet or a diet containing 5 percent black-raspberry powder. During the third week, half the animals in each diet group were injected three times with a chemical carcinogen, N-nitrosomethylbenzylamine. The animals continued consuming the diets during the week of carcinogen treatment.

After the third week, the researchers examined the animals’ esophageal tissue, thereby capturing gene changes that occur early during carcinogen exposure. Their analyses included measuring the activity, or expression levels, of 41,000 genes. In the carcinogen-treated animals, 2,261 of these genes showed changes in activity of 50 percent or higher.

“These changes in gene expression correlated with changes in the tissue that included greater cell proliferation, marked inflammation, and increased apoptosis,” Stoner says.

In the animals fed berry powder, however, a fifth of the carcinogen affected genes – exactly 462 of them – showed near-normal levels of activity, when compared with controls. Most of these genes are associated with cell proliferation and death, cell attachment and movement, the growth of new blood vessels and other processes that contribute to cancer development. The tissue also appeared more normal and healthy.

Lastly, of the 462 genes restored to normal by the berries, 53 of them were also returned to normal by a second chemoprevention agent tested during a companion study.

“Because both berries and the second agent maintain near-normal levels of expression of these 53 genes, we believe their early deregulation may be especially important in the development of esophageal cancer,” Stoner says.

“What’s emerging from studies in cancer chemoprevention is that using single compounds alone is not enough,” Stoner says. “And berries are not enough. We never get 100 percent tumor inhibition with berries. So we need to think about another food that we can add to them that will boost the chemopreventive activities of berries alone.”

Funding from the National Cancer Institute supported this research.

Influenza Antibody Spray Success: Opens New Approach to Respiratory Disease Control

Influenza Antibody Spray Success: Opens New Approach to Respiratory Disease Control

MELBOURNE, Australia & NEW YORK  August 21, 2008 -- Anadis Limited (ASX: ANX), a research driven biopharmaceutical company focused on polyclonal antibodies to address human disease, announced today important progress concerning its accelerated program to develop a nasal antibody spray to provide immediate post-exposure prophylaxis against a range of influenza viruses.

This work supports an aggressive commercial strategy expanding our intellectual property position in this unique area. Our approach shows for the first time a cost effective method of fighting influenza using antibodies. We plan to move on our own and with partners to bring this revolutionary product to market.

Working with an animal influenza challenge model in the laboratory of Professor Loreena Brown at the Department of Microbiology and Immunology, University of Melbourne, scientists successfully completed a series of proof-in-principal experiments. These experiments using an animal model designed specifically to test influenza prevention and treatment, demonstrated the ability of Anadis’ all natural antibody nasal spray formulation to “switch-off” virus replication at the respiratory tract lining. The studies indicated a significant impact of single and daily dose of the Anadis polyclonal antibodies in reducing the likelihood of infection and reducing the severity of those infected with influenza.

Scientists from the University of Melbourne and Anadis teams are in the process of submitting data for publication and intend to present the details at an upcoming influenza control conference.

Anadis CEO Dr. Zeil Rosenberg stated, “Our new topical spray approach to protecting individuals during seasonal or epidemic flu outbreaks is a radical new paradigm to protect people from these influenza viruses. The fact that the Anadis antibodies are all natural and topically applied means we may have a very safe and effective new approach to provide immediate protection to all persons and be able to address multiple virus strains. We know from current failures in vaccine protection among many persons this past year that indeed novel approaches like this are urgently needed.”

Anadis VP for Business Development Oren Fuerst further noted, “This work supports an aggressive commercial strategy expanding our intellectual property position in this unique area. Our approach shows for the first time a cost effective method of fighting influenza using antibodies. We plan to move on our own and with partners to bring this revolutionary product to market.”

Anadis’ polyclonal antibodies are obtained using its own proprietary approach to vaccinating dairy cows and then leveraging advanced dairy industry methods collect first-milking colostrum under GMP conditions, where it is further formulated as a spray dried powder, for later reconstitution as an all natural nasal spray.

Lick it, Lick it Good. Saliva Compound shown to speed healing.

Licking your wounds: scientists isolate compound in human saliva that speeds wound healing
New research in The FASEB Journal "raises expectorations" for people with chronic wounds

Bethesda, MD—A report by scientists from The Netherlands published online in The FASEB Journal (http://www.fasebj.org) identifies a compound in human saliva that greatly speeds wound healing. This research may offer hope to people suffering from chronic wounds related to diabetes and other disorders, as well as traumatic injuries and burns. In addition, because the compounds can be mass produced, they have the potential to become as common as antibiotic creams and rubbing alcohol.

“We hope our finding is ultimately beneficial for people who suffer from non-healing wounds, such as foot ulcers and diabetic ulcers, as well as for treatment of trauma-induced wounds like burns,” said Menno Oudhoff, first author of the report.

Specifically, scientists found that histatin, a small protein in saliva previously only believed to kill bacteria was responsible for the healing. To come to this conclusion, the researchers used epithelial cells that line the inner cheek, and cultured in dishes until the surfaces were completely covered with cells. Then they made an artificial wound in the cell layer in each dish, by scratching a small piece of the cells away. In one dish, cells were bathed in an isotonic fluid without any additions. In the other dish, cells were bathed in human saliva. After 16 hours the scientists noticed that the saliva treated “wound” was almost completely closed. In the dish with the untreated “wound,” a substantial part of the “wound” was still open. This proved that human saliva contains a factor which accelerates wound closure of oral cells. Because saliva is a complex liquid with many components, the next step was to identify which component was responsible for wound healing. Using various techniques the researchers split the saliva into its individual components, tested each in their wound model, and finally determined that histatin was responsible. 

“This study not only answers the biological question of why animals lick their wounds,” said Gerald Weissmann, MD, Editor-in-Chief of The FASEB Journal, “it also explains why wounds in the mouth, like those of a tooth extraction, heal much faster than comparable wounds of the skin and bone. It also directs us to begin looking at saliva as a source for new drugs.”

This article is scheduled for final publication in the November 2008 issue of The FASEB Journal (http://www.fasebj.org), which is published by the Federation of American Societies for Experimental Biology (FASEB) and is the most cited biology journal worldwide according to the Institute for Scientific Information. FASEB comprises 21 nonprofit societies with more than 80,000 members, making it the largest coalition of biomedical research associations in the United States. FASEB advances biological science through collaborative advocacy for research policies that promote scientific progress and education and lead to improvements in human health.

End in sight for the dreaded dentist drill

Dentist drill may become a thing of the past thanks to new treatment using Ramon spectroscopy being developed at Kings College London

A new technology that spots tooth decay almost as soon as it's begun promises to reduce the need for drilling and filling, writes Patrick Walter in SCI's Chemistry & Industry (C&I) magazine.

Drilling is one of the top dental phobias and puts thousands of people off visiting their dentist every year.

The new technology, which may be available in dental surgeries in five years from now, is based on Raman spectroscopy most commonly used to distinguish between different chemicals by identifying each molecule's unique fingerprint. It detects decay simply and painlessly by pointing a tiny optical fibre at the tooth to check on its health.

A preliminary study at King's College London, where the technique is being developed, found that chemical changes in the tooth could be detected by analysing how light is scattered when a laser is fired at the tooth. Researchers were able to tell healthy teeth from carious teeth because bacteria, responsible for the decay, scatter light in a different way to healthy teeth. The results were presented at Microscience 2008.

Frances Downey, a PhD student working on developing the technique at King's College London, said: 'The earlier you spot decay the better as you can remineralise the area so there is no cavitation and therefore no need for a filling.'

Dr Frederic Festy, who is supervising the project, is planning a larger trial using more teeth samples and hopes to move onto human trials soon. The key to the technique is its simplicity, he explains.

Currently, decaying teeth are uncovered either by visual examination or the use of x-rays, but usually by then, the damage has been done and the decayed area must be drilled out. But Dr Steven Hogg, a microbiologist at Newcastle University's dental school, confirms that it is possible to repair teeth with a special mouthwash or fluoride varnish if dental decay is caught early enough.

The downside of developing the machines is the cost and the time it takes to do a scan – 30 seconds can be a long time for any patient to remain perfectly still.

Leading Plastic Surgeons Reveal Revolution in Stem Cell Research Has its Roots in Beverly Hills Plastic Surgery

Leading Plastic Surgeons Reveal Revolution in Stem Cell Research Has its Roots in Beverly Hills Plastic Surgery

The latest developments in adipose stem cell research were not discovered in a Petri dish or in a lab. Instead this promising revolution in medicine was inspired via unexpected benefits resulting from facial fat grafting, a popular procedure developed and perfected by renowned Beverly Hills Plastic Surgeon Dr. Richard Ellenbogen. Stem cell researcher and former student of Dr. Ellenbogen's, Dr. Peter Rubin is now leading the latest developments in this most promising field of medicine.

Beverly Hills, CA (PRWEB) July 8, 2008 -- Dr. Richard Ellenbogen is considered the father of fat grafting, but he never dreamed that a procedure he began pioneering in the late 1970's would inspire a whole new revolution in modern stem cell research. Initially developed as a natural filler for patients' faces, facial fat grafting is a technique commonly used by plastic surgeons to replace lost volume, erase facial lines, and highlight facial features using the patient's own fat, or "adipose" tissue harvested via liposuction.

Since its creation, fat grafting has often been criticized for not lasting because the body absorbs the fat. But, with over 3,500 fat grafting procedures, Dr. Ellenbogen has added many nuances to his technique to make the fat stay put. "Fat grafting is completely technique dependent, doctors not following the proper principles might as well inject salt water," revealed Dr. Ellenbogen. "Stem cells are in the fat and have always been in the fat. The reparative results are seen in weeks and seem to improve with time," said Ellenbogen.

Fat grafting is completely technique dependent, doctors not following the proper principles might as well inject salt water

Over the last 25 years Dr. Ellenbogen and other clinicians have performed thousands of facial fat grafting procedures. They noticed several benefits of fat grafting on the immediately surrounding tissue that were not easily explained by volume replacement alone. As the patient heals from fat grafting, doctors noticed that fat placed next to bone seemed to take on the characteristics of bone, and fat placed into the muscle began to take on the characteristics of muscle. "In fact, it was and is consistently observed that the transplanted fat did not feel like isolated collections of fat, but remarkably, rather more like the recipient-site tissue into which it had been transplanted," said Ellenbogen.

Enter Dr. Peter Rubin, a co-founder of the International Federation of Adipose Therapeutics and Science. "The work we're doing today was very much inspired by the successful history of fat grafting by plastic surgeons such as Dr. Ellebogen," said Dr. Rubin. "We are now taking these adipose stem cells and proving that they can learn to be just about any type of tissue in the human body including skin, bone, muscle, nerve, liver and cardiac (heart) muscle."

The research is revealing major implications for the future of reconstructive and plastic surgery as well as the world of medicine at large. "We are looking to repair body parts lost to cancer and even re-generate parts for soldiers and accident survivors. The research is really promising," concluded Dr. Rubin.

Today, it is known that there are approximately one million stem cells in two teaspoons of fat. While there are techniques being employed by surgeons in the U.S. to enrich the stem cells in the fat, other companies are using technology in Europe and Asia to harvest pure concentrations of stem cells directly from the fat. The harvested stem cells are remixed with a smaller portion of fat resulting in a graft that is "supercharged" with stem cells. It is hoped that this supercharged graft will enhance results and broaden the applications.

About Dr. Peter Rubin:
Dr. Rubin is assistant professor of plastic and reconstructive surgery at the University of Pittsburgh School of Medicine and is a co-founder of the International Federation of Adipose Therapeutics and Science. He is a member of the Alpha Omega Alpha Society, Diplomate of the American Board of Surgery (2000), Diplomate of the American Board of Plastic Surgery (2004), and Fellow of the American College of Surgeons (2005). Dr. Rubin was recently selected to receive a Presidential Early Career Award for Scientists and Engineers (PECASE). Dr. Rubin was recently featured in a Live Science article discussing the future of adipose stem cells.

About Dr. Richard Ellenbogen:
Dr. Ellenbogen is founder of the prestigious Beverly Hills Body Plastic Surgery Specialists. As one of the most published and innovative plastic surgeons, many of the techniques Dr. Ellenbogen has helped create are used and referred to by other plastic surgeons. A two-time recipient of the American Medical Association Physicians Recognition Award, Dr. Ellenbogen is a member of the American Society of Plastic Surgery, the American Aesthetic Plastic Surgery Society, a Fellow of the American College of Surgeons, and a Fellow of the International College of Surgeons.

Herpes Cure On The Way, Free Sex For Everyone!

DURHAM, N.C. – Now that Duke University Medical Center scientists have figured out how the virus that causes cold sores hides out, they may have a way to wake it up and kill it.

Cold sores, painful, unsightly blemishes around the mouth, have so far evaded a cure or even prevention. They're known to be caused by the herpes simplex virus 1 (HSV1), which lies dormant in the trigeminal nerve of the face until triggered to reawaken by excessive sunlight, fever, or other stresses.

"We have provided a molecular understanding of how HSV1 hides and then switches back and forth between the latent (hidden) and active phases," said Bryan Cullen, Duke professor of molecular genetics and microbiology.

His group's findings, published in Nature, also provide a framework for studying other latent viruses, such as the chicken pox virus, which can return later in life as a case of shingles, and herpes simplex 2 virus, a genitally transmitted virus that also causes painful sores, Cullen said.

Most of the time, HSV1 lives quietly for years, out of reach of any therapy we have against it. It does not replicate itself during this time and only produces one molecular product, called latency associated transcript RNA or LAT RNA.

"It has always been a mystery what this product, LAT RNA, does," Cullen said. "Usually viral RNAs exist to make proteins that are of use to the virus, but this LAT RNA is extremely unstable and does not make any proteins."

In studies of mice, the team showed that the LAT RNA is processed into smaller strands, called microRNAs, that block production of the proteins that make the virus turn on active replication. As long as the supply of microRNAs is sufficient, the virus stays dormant.

After a larger stress, however, the virus starts making more messenger RNA than the supply of microRNAs can block, and protein manufacturing begins again. This tips the balance, and the virus ultimately makes proteins that begin active viral replication.

The new supply of viruses then travels back down the trigeminal nerve, to the site of the initial infection at the mouth. A cold sore always erupts in the same place and is the source of viruses that might infect another person, either from direct contact, or sharing eating utensils or towels, Cullen said.

The approach to curing this nuisance would be a combination therapy, Cullen said. "Inactive virus is completely untouchable by any treatment we have. Unless you activate the virus, you can't kill it," he said.

Cullen and his team are testing a new drug designed to very precisely bind to the microRNAs that keep the virus dormant. If it works, the virus would become activated and start replicating.

Once the virus is active, a patient would then take acyclovir, a drug that effectively kills replicating HSV1.

"In principle, you could activate and then kill all of the virus in a patient," Cullen said. "This would completely cure a person, and you would never get another cold sore."

He and the team are working with drug development companies in animal trials to begin to answer questions about how to deliver this drug most effectively.

Co-authors included Jennifer Lin Umbach, Ph.D., and Heather W. Karnowski, B.S., of the Duke Department of Molecular Genetics and Microbiology and Center for Virology, and Martha F. Kramer, Igor Jurak, and Prof. Donald M. Coen of the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. This work was supported by two NIH grants.

HIV DNA therapy permanently locks out the virus from cells creating a permanent 10 fold suppression of the virus.

Mice have been made resistant to HIV by sabotaging a gene in the blood cells that the virus normally infects.

Researchers who developed the treatment at Sangamo BioSciences, a biotechnology company in Duarte, California, US, hope to test it in patients by the end of 2008. If successful, the treatment could offer a more effective way for controlling HIV in patients with the disease, the researchers say.

Once the gene has been altered, the cells can no longer make CCR5, a surface protein to which the virus attaches itself before sneaking inside. With no "door handle" to hold onto, the virus can no longer infect the cell.

To sabotage the gene that makes CCR5, they used a harmless virus to sneak a molecule called a zinc-finger nuclease into the cells.

Permanent change

The "zinc-finger" part of the molecule targets and binds exclusively to genetic material found only in the CCR5 gene. The "nuclease" section is an enzyme that snips open and alters the gene so that it can no longer make the CCR5 protein.

After this process, the cell is then effectively immune to infection by HIV. "Once the job is done, the cell DNA is altered permanently," says Elena Perez at the Children’s Hospital of Philadelphia in Pennsylvania, who collaborated on the study.

Half the mice received human T-cells treated with the zinc-finger nucleases, and the other half received untreated T-cells. Later the mice were infected with HIV.

Inherited trait

After six weeks, all the treated mice had become resistant to the virus. "We saw a tenfold suppression of the virus in the treated mice compared with controls," says Philip Gregory of Sangamo, "and we saw a five-fold increase in the number of circulating T-cells, [which are] usually attacked by HIV."

In human trials, Sangamo plans to extract T-cells from HIV-infected patients, treat them in the lab to alter the gene that makes CCR5, then return them to the bloodstream.

The hope is that because these cells are resistant to HIV, as they multiply they will become the dominant type within the body. They could then provide longer-term protection than drugs that deny HIV access to cells by physically blocking CCR5 molecules.

"What's really exciting is that the change in the genome is permanent, and inherited by all 'daughter' T-cells created when the altered T-cells multiply," says Gregory.

Drug fallback

"The zinc-finger approach has significant potential compared to other strategies," says Ed Berger, a researcher credited with helping establish the CCR5-HIV link at the National Institutes of Health in Bethesda, Maryland, US.

"With genetic knockout of CCR5 by the zinc finger, the cells lacking CCR5 have a selective advantage," he says.

Berger adds that, unlike other approaches where patients have to carry on taking CCR5 blockers, or which depend on molecules that must continuously stop CCR5 working, the zinc finger only has to do one operation, and the job is complete.

John Moore, co-discoverer of the link between CCR5 and HIV in 1996, says the science is excellent, but doubts whether the gene can be sabotaged in enough T-cells to make a difference to patients.

He says that in any case, Maraviroc – a CCR5 blocking drug launched last year by Pfizer in the US and Europe – works well, and that others are in advanced clinical trials.

Cancer treatment found that cures 100% of every cancer in mice to be tested in humans.

Granulocytes go to work on removing human cancerous cells.

'All the mice we treated were 100 per cent cured' lead researcher Dr. Zheng Cui told CTV News. 'So that was very surprising for us.'

Scientists to test if cancer cure can work in humans

American researchers will soon start a human trial to determine whether a treatment that can eradicate cancer in mice will do the same in people.

The treatment will transfuse specific white blood cells, called granulocytes, into patients with advanced forms of cancer. The granulocytes will come from healthy young people with immune systems that produce cells that have high levels of anti-cancer activity.

In the animal studies, white blood cells from cancer-resistant mice cured all lab mice who had malignant tumours. The cells have also been able to kill cervical, prostate and breast cancer tumour cells in Petri dish tests.

"All the mice we treated were 100 per cent cured," lead researcher Dr. Zheng Cui told CTV News. "So that was very surprising for us."

Cui, an associate professor of pathology at Wake Forest University Baptist Medical Center in North Carolina, will announce the study Saturday at the Understanding Aging conference in Los Angeles.

Granulocytes account for about 60 per cent of all white blood cells in the human body. The scientists already know, via a small study of human volunteers, that granulocytes from people under the age of 50 are most effective at killing cancer cells.

The study will begin with 22 cancer patients for whom conventional treatment has been unsuccessful. The researchers say that they will know within three months if the treatment will work in humans.

Cancer researchers worldwide will be watching the tests closely.

"Certainly in the mouse, being able to do these things is quite remarkable and very exciting," said Dr. Ronan Foley of the Juravinski Cancer Centre in Hamilton, Ont. "Oftentimes when it is translated into the human situation it doesn't work as well. But that doesn't mean it isn't going to work."

Spray-on skin gun will make healing wounds a much faster affair (w/cool pic)

Spray-on skin gun will make healing wounds a much faster affair

The process of healing a wound isn't a short one, and it isn't a fun one, especially if it's a more serious wound. You've got to keep it clean and bandaged, and it'll hurt for a while and take a good amount of time to actually heal. That's no fun. We're living in the 21st century, we shouldn't have to put up with that sort of crap.

Scientists are working on a gun that sprays new, stem cell infused skin onto a wound, essentially speeding up the healing process to a matter of hours. The Armed Forces Institute for Regenerative Medicine just invested $250 million in researching advancements such as these that'll make healing and fixing damaged body parts a snap, so hopefully it'll only be a matter of time before losing a few inches of skin will only be a minor inconvenience.

Encouraging results in experiments on mice are raising hopes of an effective colon cancer vaccine

A protein found only in the intestines may help lead the way to a vaccine that can treat colon cancers and perhaps other tumors too, U.S. researchers reported on Tuesday.

A flu shot is administered. Researchers have taken a step toward developing a vaccine for colorectal cancer.  

A nurse fills a syringe with flu vaccine at the Arkansas State Fair Grounds. Scientists say a vaccine for colon cancer may be possible in the future. 

Dr. William Taylor, medical director of colorectal surgical oncology for the John B. Amos Cancer Center, looks over scans in his office in Columbus, Ga. 

Mice immunized with the protein and then infected with colon tumors had fewer tumors spread to the lung and liver than usual, they reported in the Journal of the National Cancer Institute.

Many cancer vaccines are in the works but researchers are struggling to find the right targets, called antigens, that are found only on tumors and not in healthy tissue.

Adam Snook and Dr. Scott Waldman of Thomas Jefferson University in Philadelphia and colleagues decided to look at colon cancer in particular because the intestinal lining and some other so-called mucosal areas are protected from much of the activity of the immune system.

Also, some proteins from these immune-protected sites are active on tumor cells.

They looked specifically at guanylyl cyclase C protein, or GCC protein, normally only expressed, or active, in the intestinal lining and in spreading colorectal cancer cells.

The researchers injected mice with colon cancer cells before or after immunization with guanylyl cyclase C.

Unvaccinated animals had an average of 30 new tumors in the lungs and liver. Vaccinated animals had three. While such an approach does not look like a cure, it could be an important treatment, they said.

"More than 50 percent of patients with colorectal cancer die of metastatic disease, primarily in the liver and lung," they wrote. The vaccinated mice also lived longer.

Colon cancer affects an estimated 1.2 million new patients a year globally and kills 130,000.

While injecting mice with tumor cells causes cancer, it does not perfectly replicate human cancer development, researchers caution. It is far easier to treat such a mouse in the lab than a person with cancer.

But the researchers are hopeful.

"We think this identifies a novel class of vaccine candidate targets for tumors that originate and metastasize from mucosa, like colorectal cancer," Waldman said in a statement.

The approach may also work in cancers of the head and neck, lung, breast, vagina and bladder, all of which begin in the mucosa, they said.

Cancer vaccines that have shown more promise include one made by Antigenics Inc against melanoma, the deadliest form of skin cancer, and another made by Avant Immunotherapeutics Inc and licensed by drug giant Pfizer Inc that attacks glioblastoma multiforme, the most common and deadly type of brain tumor.

Cuban scientists said on Tuesday they had made available a vaccine that extends the lives of lung cancer patients.

‘Elephant Legs’ Woman Finds Hope Outside of China

http://www.weirdasianews.com/2008/06/15/elephant-legs-woman-china/

‘Elephant Legs’ Woman Finds Hope Outside of China

Wang Cheng, a 24 year-old Chinese girl often referred to as ‘Elephant Legs’, has a rare medical condition of elephantiasis, which has caused her legs to swell nearly three times their normal size.

Wang has suffered from the condition since the age of 6 and currently cannot even wear pants because of the pain it causes her 110 pound legs.

“I cannot go out to work. Nor can I wear trousers as normal people.” Wang said while sitting in her bed at Taipei Municipal Wan Fang Hospital waiting for an upcoming operation.

Living with a paralyzed father and an unemployed mother in China, Wang was given a blessing when she was offered an opportunity to receive free surgery that would cut away some of the lymphatic tissue in her legs.

Hsu Wen-hsien, a senior doctor who will lead the operation on Monday, indicated that he had never seen such a sever case in all his 30 years experience.

“In the past cases, none of the patients have had two legs swelling to this size,” said Hsu.

The operation is expected to reduce Wang’s leg size by about 35% initially, and up to 50% after about 2 months of rehabilitation.

Taiwanese Buddhist group Fo Kuang Shan is paying for the travel expenses to Taiwan and non-medical expenses while the 10-hour operation and related care, estimated at $17,600, is being provided free by the hospital.

Elephantiasis, or lymphatic filariasis, is often transmitted by mosquitoes.

Snorers should be tested for diabetes

SNORE a lot? Maybe you have diabetes. People with sleep apnoea, who briefly stop breathing and snort themselves awake, often also have type 2 diabetes - and vice versa. In fact, the evidence of a link is so strong that the International Diabetes Federation (IDF) is urging doctors treating patients with one condition to test for the other.

"There's no question about the link," says Paul Zimmet, co-chairman of the IDF's task force on epidemiology and prevention, based in Melbourne, Australia. The IDF says that 40 per cent of patients with apnoea have diabetes, while about 23 per cent of diabetics have apnoea (Diabetes Research and Clinical Practice, DOI: 10.1016/j.diabres.2008.04.025).

Identifying both diseases saves lives, says Zimmet. Sleep apnoea is the commonest treatable cause of both high blood pressure and heart failure. Meanwhile, type 2 diabetes can also be fatal but is easily managed with the right diet and insulin.

The race is now on to identify all patients who have both and to investigate the cause of the link.

Man Feels Fine After Being Shot In Head By Nailgun - Used a Common Claw Hammer to Remove

George Chandler said he feels fine, even though a nailgun fired a 2.5 inch nail into the top of his head on Friday. Chandler and a friend were doing a project in a backyard when the nailgun hose became tangled, causing the tool to fire one nail.

Chandler said Monday he told his friend he didn't know where the nail went, but he felt a sting on the top of his head.

They discovered that the nail was driven deep into Chandler's head, so they called an ambulance and he was rushed to a hospital.

Chandler said a doctor used a common claw hammer to remove the nail.

He said he feels "very lucky, very, very lucky" to have escaped serious injury.

Brain Stem Cells Can Be Awakened, Say Scientists.

Scientists at Schepens Eye Research Institute have identified specific molecules in the brain that are responsible for awakening and putting to sleep brain stem cells, which, when activated, can transform into neurons (nerve cells) and repair damaged brain tissue. Their findings were recently published online in the Proceedings of the National Academy of Science (PNAS)

An earlier paper (published in the May issue of Stem Cells) by the same scientists laid the foundation for the PNAS study findings by demonstrating that neural stem cells exist in every part of the brain, but are mostly kept silent by chemical signals from support cells known as astrocytes.

"The findings from both papers should have a far-reaching impact," says principal investigator, Dr. Dong Feng Chen, who is an associate scientist at Schepens Eye Research Institute and an assistant professor of ophthalmology at Harvard Medical School. Chen believes that tapping the brain's dormant, but intrinsic, ability to regenerate itself is the best hope for people suffering from brain-ravaging diseases such as Parkinson's or Alzheimer's disease or traumatic brain or spinal cord injuries.

Until these studies, which were conducted in the adult brains of mice, scientists assumed that only two parts of the brain contained neural stem cells and could turn them on to regenerate brain tissue-- the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). The hippocampus is responsible for learning and memory, while the SVZ is a brain structure situated throughout the walls of lateral ventricles (part of the ventricular system in the brain) and are responsible for generating neurons reponible for smell. So scientists believed that when neurons died in other areas of the brain, they were lost forever along with their functions.

In the first study, Chen's team learned that stem cells existed everywhere in the brain by testing tissue from different parts of adult mice brains in cultures containing support cells (known as astrocytes) from the hippocampus, where stem cells do regenerate. In the cultures the stem cells from other brain regions came to life and turned into neurons.

When they compared the chemical makeup of the areas known to generate new neurons in the hippocampus with other parts of the brain, the team discovered that astrocytes in the hippocampus were sending one signal to the stem cells and that those from the rest of the brain were sending a different signal to stem cells.

In the second (PNAS) study, the team went on to discover the exact nature of those different chemical signals. They learned that in the areas where stem cells were sleeping, atrocytes were producing high levels of two related molecules--ephrin-A2 and ephrin-A3. They also found that removing these molecules (with a genetic tool) activated the sleeping stem cells.

The team also found that astrocytes in the hippocampus produce not only much lower levels of ephrin-A2 and ephrin-3, but also release a protein named sonic hodghoc that, when added in culture or injected into the brain, stimulates neural stem cells to divide and become new neurons.

"These findings identify a key pathway that controls neural stem cell growth in the adult brain and suggest that it may be possible to reactivate the dormant regenerative potential by adding sonic hedgehoc, or blocking ephrin-A2 or ephrin-A3," says Dr. Jianwei Jiao, the first author of the two papers,

The next step for the team will be to stimulate the sleeping stem cells in animals who are models of neurodegenerative disorders, such as Parkinson's disease, to see if the brains can repair themselves and restore their damaged functions.

Japanese patient's 'tumour' turns out to be 25-year-old towel

Doctors who carried out surgery on a Japanese man to remove a "tumour" had good news and bad news for him. He did not have cancer -- but the "growth" that had been causing him pain was in fact a 25-year-old surgical towel.

The patient had been carrying the cloth since 1983, when surgeons at the Asahi General Hospital in Chiba prefecture near Tokyo left it in him after an operation to treat an ulcer, a spokesman for the hospital said.

The man, now 49, went in to another hospital in late May after suffering abdominal pain.

When examinations found what was believed to be an eight-centimetre (3.2-inch) tumour, he underwent the operation to remove it. It was only then that surgeons realised it was a towel.

"The towel was greenish blue although we are not sure about its original colour," the Asahi General Hospital spokesman said, adding it had been crumpled to the size of a softball.

Asahi hospital officials visited the man and apologised, he said.

The former patient has no plans to sue the hospital, which is in talks with him over compensation or other measures, the official said.

Japanese media reports said the man, who was not identified, still had his spleen removed.