Molecular Pathology of the hMSH2 Mutator Gene and Its Transcripts in Patients With Colorectal Cancer in the West of Scotland

Colorectal cancer (CRC) is the second or third most common cancer in Western countries, and its incidence is rising. Among the hereditary forms Familial Adenomatous Polyposis (FAP) is a rare, dominantly inherited disease which is caused by germ-line mutations of the Adenmatous Polyposis Colorectal Cancer (APC) gene. A second form of CRC that shows familial aggregation is Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The main aims of this study were: (i) to assess microsatellite instability in the tumour of patients with colorectal carcinoma and (ii) to identify germ-line mutations in two mismatch repair genes, using a variety of techniques. To do this, in two groups of unselected patients, 30 pairs of normal/tumour DNAs and 47 RNA samples were made available for microsatellite instability studies and mutation analysis, respectively. Nine polymorphic markers were chosen for microsatellite instability analysis including: D13S160, D2S119, D8S282, D18S34, 635/636, D2S123, LPLCA- A, D2S378, LPLCA, B. Using this approach, 30% (9/30) of the tumours exhibited instability at one or more loci (RER+). There was no significant difference between age and sex in patients with or without microsatellite instability. Using the reverse transcriptase polymerase chain reaction (RT-PCR), the entire coding sequence of the hMSH2 gene was amplified in either two or three overlapping segments. By employing RNA and DNA based techniques a number of germ-line mutations were found in the hMSH2 and hLMH1 genes. The mutations included: one novel splice site mutation responsible for exon 15 skipping, two novel deletions of exons 2-6 and 2-8 from the mRNA transcripts, four different mRNA deletions at different sites in the hMSH2 gene, one missense mutation at exon 6 and three intronic mutations at positions +9, -10 and +19 in intron 1, 11, and 14 respectively. A variation of the published sequence was also found at 3' end of the untranslated region in the hMSH2 gene. None of the families in this study were large enough for linkage analysis. In one HNPCC family, one splice site mutation was found at position +5 in intron 15. This resulted in exon 15 skipping from transcripts and produced a truncated hMSH2 protein. This germ-line mutation was present in 4 affected members of the family, but was not present in the healthy members of the family or in 177 other colorectal cancer patients. The individual who bears this germ-line mutation showed a RER+ phenotype in the tumour sample. A patient whose tumour was RER+ had a deletion of exons 2 to 6 inclusive which resulted in an out-of- frame deletion of codons 71-359. This deletion created a premature termination codon within exon 7 at nucleotide position +3 from the splice site. No genomic mutations were found which could account for the exon skipping. No other member of the family was available for further investigation. A patient with a tumour showing the RER+ phenotype had a deletion of exons 2 through exon 8 inclusive which resulted in an out-of- frame deletion of codons 71-466. This deletion also created a premature termination codon within exon 9 at position +12 from the splice site. No genomic alterations were found which could account for the exon skipping. This individual was the only member of the family available for investigation. Four different partial mRNA deletions in different exons were found. Three of them produced premature termination codons. No alteration could be found by sequencing the genomic splice sites, or in the corresponding exons. One of the partial deletions was found in four unrelated individuals. The breakage points of the aberrant mRNAs were not located at the splice site junctions in any case. In order to clarify whether or not these partial mRNAs deletions are normal, RT-PCR from 20 healthy individuals was carried out. The whole coding sequences of the hMSH2 gene were amplified in three overlapping fragments and no extra bands were observed. These results indicate that none of the mRNA deletions reported in this study are common normal variations. A T to C transition was found in a short polypyrimidine tract in intron 12 at nucleotide position -6. Particularly, short poylypyrimidine tracts at the 3' end of the intron have been shown to effect mRNA splicing (Roscigno et al. 1993), but this mutation had no affect on the mRNA splicing process, since no short transcript was found. It was, however, found in 15% of the normal population when 86 normal chromosomes were tested. A G to A transition was also found at codon 322 in exon 6 which changes glycine to asparatic acid. Since glycine is a non- polar neutral amino acid and asparatic acid is an acidic one, it is likely that the polarity of the protein will be changed

Chloroplast-Derived Vaccine Antigens Confer Dual Immunity Against Cholera and Malaria by Oral or Injectable Delivery

Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10+ T cell but not Foxp3+ regulatory T cells, suppression of interferon-γ and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (\u3e300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity

The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice

<div><h3>Background</h3><p>Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.</p> <h3>Materials and Methods</h3><p>Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.</p> <h3>Results</h3><p>AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.<em>Scid</em> mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.</p> <h3>Conclusion</h3><p>The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.</p> </div

The Green Vaccine: A Global Strategy To Combat Infectious And Outoimmune Diseases

Plant derived oral green vaccines eliminate expenses associated with fermenters, purification, cold storage/transportation and sterile delivery. Green vaccines are expressed via the plant nuclear or chloroplast genomes. Chloroplast expression has advantages of hyper-expression of therapeutic proteins (10,000 copies of transgene per cell), efficient oral delivery and transgene containment via maternal inheritance. To date, 23 vaccine antigens against 16 different bacterial, viral or protozoan pathogens have been expressed in chloroplasts. Mice subcutaneously immunized with the chloroplast derived anthrax protective antigen conferred 100% protection against lethal doses of the anthrax toxin. Oral immunization (ORV) of F1-V antigens without adjuvant conferred greater protection (88%) against 50-fold lethal dose of aerosolized plague (Yersinia pestis) than subcutaneous (SQV) immunization (33%). Oral immunization of malarial vaccine antigens fused to the cholera antigen (CTB-AMA1/CTB-Msp1) conferred prolonged immunity (50% life span), 100% protection against cholera toxin challenge and inhibited proliferation of the malarial parasite. Protection was correlated with antigen-specific titers of intestinal, serum IgA & IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. High level expression in edible plant chloroplasts ideal for oral delivery and long-term immunity observed should facilitate development of low cost human vaccines for large populations, at times of outbreak. © 2009 Landes Bioscience

The Green Vaccine: A Global Strategy to Combat Infectious and Autoimmune Diseases

Plant derived oral green vaccines eliminate expenses associated with fermenters, purification, cold storage/transportation and sterile delivery. Green vaccines are expressed via the plant nuclear or chloroplast genomes. Chloroplast expression has advantages of hyper-expression of therapeutic proteins (10,000 copies of trans-gene per cell), efficient oral delivery and transgene containment via maternal inheritance. To date, 23 vaccine antigens against 16 different bacterial, viral or protozoan pathogens have been expressed in chloroplasts. Mice subcutaneously immunized with the chloroplast derived anthrax protective antigen conferred 100% protection against lethal doses of the anthrax toxin. Oral immunization (ORV) of F1-V antigens without adjuvant conferred greater protection (88%) against 50-fold lethal dose of aerosolized plague (Yersinia pestis) than subcutaneous (SQV) immunization (33%). Oral immunization of malarial vaccine antigens fused to the cholera antigen (CTB-AMA1/CTB-Msp1) conferred prolonged immunity (50% life span), 100% protection against cholera toxin challenge and inhibited proliferation of the malarial parasite. Protection was correlated with antigen-specific titers of intestinal, serum IgA & IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. High level expression in edible plant chloroplasts ideal for oral delivery and long-term immunity observed should facilitate development of low cost human vaccines for large populations, at times of outbreak

AG490 prevents development of autoimmune diabetes.

<p>Prediabetic female NOD mice at 4 (UT: n = 11; DMSO: n = 13; and AG490 treated mice: n = 15 mice/group) (A), or at 8 week of age, (n = 10 per group) (B) were treated with AG490 or DMSO (i.p) for 5 consecutive week, three time per week followed by once a week for 5 more week [Kaplan-Meier test (*p = 0.02, 1A) and (**p = 0.005, 1B, at week 30)]. Our data also indicate that significant differences (p<0.01) was observed between the AG490 and DMSO treated mice for up week 52; when treatment was started at week 4. Body weight of NOD mice treated with AG490 (C) or DMSO (D) in mice when treatment initiated from 4 weeks of age is shown. [This figure is intended to be in color online and black and white in print.]</p

AG490 does not block infiltration of leukocytes in the islets.

<p>A- Prediabetic NOD mice at 4 weeks of age were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive weeks (3× per week) and mice were sacrificed one week after the last injection at week 10. Insulitis was scored by two different investigators and an average of two different scores is shown (p = n.s). At least 150 islets per group in two interrupted sections, 100 µm apart, was scored. B- The C-peptide level was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036079#s4" target="_blank">materials and methods</a> and no significant differences were found. C- Five prediabetic NOD mice at 4- weeks of age were treated with AG490 or DMSO (n = 5 per group) as described above for 5 consecutive weeks and then sacrificed at week 10, C- real-time RT-PCR was performed on enriched infiltrated lymphocyte from the pancreas of 5 mice/group and expression level of Foxp3 and TGF-β1 was measured in three independent RT-PCR by TaqMan Gene expression (Life Technologies, Applied Biosystems, Assays-on-Demand) (* = expression was very low and/or undetectable). [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036079#pone-0036079-g004" target="_blank">Figure 4A</a> is intended to be in color online and black and white in print.]</p

AG490 modulates phenotype and function of DC.

<p>A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036079#s4" target="_blank">materials and methods</a> and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO <i>in vivo</i>. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC <i>in vitro</i> as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated <i>in vitro</i> with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×10⁵) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of ³H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student <i>t</i>-test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are intended to be in color online and black and white in print].</p

AG490 induces remission of diabetes in NOD and in STZ-induced diabetic mice.

<p>Newly diagnosed and established diabetic NOD mice were treated with AG490 (i.p, 1 mg/mouse) without insulin supplement (A) (n = 23) or with DMSO (B) (n = 10). Blood glucose was measured by ACCU-CHECK 2–3 times weekly. Sustainable euglycemia was observed for several months after drug withdrawal while DMSO treated mice expired within 2–4 week after disease onset. The average blood glucose at the disease onset in the cured NOD mice was 314.43 mg/d (Max = 374; Min = 277 mg/dl) versus 384.58 mg/dl (Max = 478, Min = 335 mg/dl) in mice that AG490 failed to restore euglycemia (p<0.0014). An average blood glucose in cured NOD mice treated with AG490 was 169.82 mg/dl±23.21, n = 7 versus 555.13±63.79, n = 17 in uncured NOD mice (p = 1.15E-11). (C) Diabetes was induced by STZ injection in male BALB/c mice and one week after the last injection diabetic mice were treated with either AG490 or DMSO (D) (n = 5/group). The blood glucose level was significantly lower (p<0.004) in AG490 treated mice when compared with DMSO treated mice. [This figure is intended to be in color online and black and white in print.]</p