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|968 sq. ft.
|Property type
|Minimum Stay
|3 nights
The Florentine Retreat is a house of 90 sqm with garden and terrace .It is possible to rent only a suite for two people.
It consists of 2 apartments of 45 sqm each, in a Villa with 1 double bedroom each, a living room, 2 bathrooms with original Amalfi tiles ( with showers), 2 kitchnette: 1 is located in Garden apt living room with access to the garden, and 1 is located in Studio and can be used to have meals on the upper terrace during warm days.
Free Wifi and parking available.
The prices are for 2 people in one suite.
The apatment is rentable for 1-2-3-4-5-6 guests.
It's possible to add 1 single bed in the living (on request) and one in Studio..
The Suite Apartment is located in a Villa , ex Hay Loft of an old tuscan family.
The Villa is now owned by Martelli family and surrounded by gardens in a secluded area but at only 12 minutes from Forte Belvedere and Boboli garden entrance, 20 minutes from the Old Bridge and Piazzale Michelangelo; a fashinating walk in th one of the most intersting antique road of Florence. The composer Tchaikovsky loved Florence and its climate and spent a winter in a house in Via San Leonardo, less than 100 meters away from us.
When you are in this apartment you do not need a car for visiting Firenze ( historical city center is off-limits for cars)
Bus stops at 100 meters.
Anyway we have complimentary parking available.
The owner is glad to help you to book museum tickets
You can also do it by yourself calling the booking office: the Uffizi Gallery is 0039.055.294833; this number is also valid for the other state museums ) or at www.polomuseale/firenze
There are many temporary exhibitions held in Florence throughout the year. Some of great interest include the bi-annual antiques show in October, the Craftsmanship's Exhibition and the Maggio Musicale Fiorentino's season of concerts and operas. In some seasons, it is possible to observe the night sky from the nearby Arcetri's Observatory of Galilei.
A local Open Market every Tuesday morning in Cascine Park,
Fashion Outlet The Mall (Armani, Gucci, Tod's, Ferragamo, Burberry.....) at Incisa Val d'Arno at 15km ( A1 motorway)
The villa is very close (100m) to the historical Chalet Fontana, caffé, restourant recentely restored with great charme, exellent cuisine, splendid park.
The Terrazza del Principe , restourant and pizzeria is at 250 m. from home, very beautiful panorama.
At the top of the hill of Arcetri, at 600m from home, there are the ancient trattoria-restaurant Omero, serving tipical tastefull tuscany food.
The Villa is located in a very beautiful area close to historical city center far from noisy and close to the best spotlights such as Piazzale Michelangelo
A garden in the city.
Dear Laura,
we are leaving today and we would like to tank you again for the perfect hospitality and your precious hints
We hope to come back next spring for a longer stay
Joan and Bill
Chateau Fontina is right down the road Laura was fantastic and accommodating!
Very nice hostesses shared all the necessary tips to make our stay an amazing experience. Our favorite remains our daily strolls down Via San Leonardo down to the Ponte Vecchio passing Tchaikovski home and through the Bardini Gardens. We will come back!
We had a wonderful stay in the Garden apartment. It was perfect for two. We had a warm welcome even though we were about 6 hours later than anticipated. Parking inside the gate was perfect, but be warned that if you have a larger car (we rented a KIA Sportage) it is a tight fit! We got a lovely welcome basket with wine, fruit, breakfast food and coffee. Bed was comfortable, bathroom was modern and good...no complaints! Everything was as promised and the walk to Ponte Vecchio was about 15-20 minutes. Would definitely return!
It was fantastic . We absolutely loved the apartment and Laura and her family were so helpful. They went out of their way for us. A lovely family. The apartment is a beautiful space filled with beautiful antique pieces and the balcony is very private and a great place to rest after a long day of walking around Florence. There is a little kitchen where we made cooked breakfast daily and dinner a couple of nights. There is a fantastic restaurant on the corner and you can walk to the Ponte de Vecchio a couple of different ways in 15 - 20 minutes. It is a great place to stay and we will be going back.
Thank you so much Laura and your lovely family
We came to Florence as part of our silver wedding anniversary celebration, and I cannot recommend Laura's apartment highly enough. It has the feel of a cosy country house with ample character and thoughtfully and tastefully decorated, with lovely rugs, terracotta tiled floor and beautiful old furniture. We absolutely loved the large French doors opening out to the pretty private garden area, surrounded by grapevine. The centre of Florence is a pleasant short walk away (around 20 mins). I believe there are also buses- but the walk is so nice!
We were met by Laura's daughter Lucia who is a lovely person and very helpful in recommending places to visit in Florence, and even took the time to talk to us about the next part of our trip to Tuscany and had some suggestions for what we could do there! Loved every minute of our stay.
We spent 4 nights in Laura´s apartment in Via San Leonardo and we were very satisfied. The place is wonderful – very quiet, elegant with a beautiful garden. We stayed at the ground-floor garden apartment, which was very comfortable also for 3 adults - well equipped, clean, nicely decorated and very calm.
Florence is a beautiful city, but with all the hustle and bustle and the swarm of tourists could be sometimes very tiring. We always appreciated this peaceful place after couple of hours in the city and we enjoyed sitting in the garden and having lunch during our midday breaks. It was very relaxing.
The apartment is located uphill from the centre. It is close to Piazzale Michelangelo and San Miniato al Monte, which offer majestic views over the city. Reaching the centre is also very easy. You can walk down and reach Ponte Vecchio in about 15-20 minutes by foot or take a bus which is about 2 minutes from the house.
This place is really very charming and Laura is a lovely person – very welcoming and careful. She greeted us with refreshment and also filled the fridge with enough breakfast food for the entire stay. The house is easily reachable from the highway and also very good located if you want to make some trips outside Florence. Laura offers private parking in the garden – this is a big bonus in Florence!
So in summary, this apartment is a perfect place in a fantastic location, with a warm and very lovely landlady Laura. We really enjoyed our stay and we would definitely recommend it to our friends. Thank you, dear Laura, we hope to come back soon!
Tanti saluti da Praga!
The Apartment, 1400m from Old Brige, is located in a beautiful Villa in Via San Leonardo, historic lane connecting the Astronomical Observatory G.Galilei on the hill of Arcetri with Forte Belvedere and Ponte Vecchio (Old Bridge).The Villa ,an ancient hay-loft, was renovated by owner and it is surrounded by a garden with vines, wisteria pergolas, laurel hedges and cypress trees.
Via San Leonardo is a narrow road through antique buildings, olive groves and gardens in the protected and silent area landscape, with few inhabitants – villas and farmhouses converted into artists' studios, that takes about twenty minutes to walk to the Old Bridge. Walking towards the Old Bridge: Chalet Fontana, historic meeting place of writers and artists, the house of Tchaikovskij, the famous painter Ottone Rosai's studio, the Church of San Leonardo in Arcetri of XI century and passing close by Forte Belvedere of XVI century.
From Forte Belvedere a gate leads into Boboli Garden and Pitti Palace, but if you go through the door of old walls, you find Bardini Museum of Macchiaioli, Galileo's home and Ponte Vecchio.
Piazzale Michelangelo is a walking distance with its marvellous view of Firenze
|Rate Period
|Weekend Night
High Season 2017
Mar 1 2017 - Nov 10 2017
3 night minimum stay
|Notes: Rates are based on 2 guests; add $53 per night, per additional guest
Winter Holiday
Dec 21 2017 - Jan 8 2018
3 night minimum stay
|Notes: Rates are based on 2 guests; add $53 per night, per additional guest
High Season 2018
Mar 1 2018 - Nov 10 2018
3 night minimum stay
|Notes: Rates are based on 2 guests; add $53 per night, per additional guest
My Standard Rate
3 night minimum stay
|Notes: Rates are based on 2 guests; add $53 per night, per additional guest
|No additional mandatory fees
|Additional Guest Fee
|$52.77 per person per night
- Prices refer to 1 full week reservation for two persons.
A 3 nights reservation only will be considered
Long Term Rental are welcome.
The charge for the added person in the living room is 150 € for week
-Prices include: accommodation for 2 persons, a welcome basket for your breakfast, electricity, water and gas for the kitchen and bathroom need. Bed linen, towels will be replaced every week.


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The MacGregors have been professional writers for 25 years. They have mined numerous synchronicities and have fruitfully used the knowledge derived from them. One synchronicity after another led to their many non-fiction books on dreams, psychic development, astrology, yoga, the tarot, divination, and animal symbolism.
They are also novelists. Rob wrote Indiana Jones and the Last Crusade and six other original Indiana Jones novels, which have sold millions of copies. He won the coveted Edgar Award for Prophecy Rock, a YA mystery, and its sequel, Hawk Moon, was an Edgar finalist. Trish, writing as T.J. MacGregor, won the Edgar in 2003 for best original paperback, for Out of Sight. Her 33 novels books have been translated into 14 languages.
We will be talking about their latest non-fiction book, Aliens in the Backyard: UFO Encounters, Abduction & Synchronicity. In it they focus on 4 abduction encounters, and the synchronicities the witnesses have begun to experience.
For more on the MacGregors and their books, visit their website at: www.synchrosecrets.com
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subset: commoncrawl


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text:
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting β cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process.
Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed.
The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D.
Our data strongly suggest that the immune related mechanisms taking place at this early age in the PLN, correlate with homeostatic changes influencing tissue integrity of the adjacent pancreatic tissue. Functional analysis of the identified genes suggested that similar mechanisms might be operating during pre-inflammatory processes deployed in tissues i) hosting parasitic microorganisms and ii) experiencing unrestricted invasion by tumour cells.
Type 1 diabetes (T1D) is an autoimmune disease characterized by the absence of insulin due to the specific destruction of the insulin-producing β cells of the pancreatic islets. This is a progressive process taking over 20 weeks in the NOD mouse and several years in human patients to be completed [1]. The NOD animal model has been a valuable source of information for several aspects of disease pathogenesis [2]. Genetic studies have contributed to portray the complexity of the disease and have established that multiple loci are carrying genes implicated in T1D in human [3] and animal models [4]. In human, more than 6 genes contribute to the disease [5,6] while over 20 loci have been described in the NOD mouse [7] but only few possible candidate genes have been unequivocally defined [8] other than the H2g7 Idd1 locus [7,9].
Despite intensive research, the initial causal events remain elusive since the selection of individual mice at early stages, prior to the overt clinical signs, represents a challenge. Indeed, even though the NOD mice are inbred not all animals develop the disease, with an incidence of 40-90% in females, depending on the colony [2]. Additional hindrances for selecting individual animals that will subsequently develop the disease with certainty are the low penetrance of the implicated genes and the influence of environmental factors. For these reasons, the exact mechanisms taking place prior to the onset of the pancreatic islet-damaging sustained inflammatory processes remain largely unknown.
The aim of our investigation was to evaluate the possibility of the existence of distinct gene expression profiles in order to eventually render possible the study of the early molecular changes, taking place before the onset of inflammation in autoimmune prone mice. One hypothesis is that during the post-weaning period in genetically autoimmune prone individuals (mouse or human), homeostatic changes prompt the immune system not to conform to physiological responses, but to instead trigger pathways that lead to the final autoimmune condition in later life. Exogenous factors including the more or less sustained presence of micro-organisms in otherwise microbe-free tissues may take place during early life stages [10]. This may influence the immune regulatory processes put in place to prevent, attenuate and/or repair the acute or sustained inflammation. It has been reported that early immune stimulation prevents autoimmune diseases, while infections later in life might exacerbate their advent in genetically prone individuals [11]. A single injection of BCG (the Mycobacterium bovis vaccine strain) is highly protective against T1D in the NOD mice [12] and BB rats [13] when administered early but it has no beneficial effect given after disease onset; similar to what has been observed in human [14]. These observations lead to the indication that the time "window" of opportunity to prevent disease in human may share similarities with the NOD mice and thus BCG or other immune-system signalling, safe biological preparation, should be given as therapy before the onset of inflammation of the pancreatic islets (insulitis). This early "window" coincides in the NOD mouse model with the early appearance of insulitis, occurring between 4 and 7 weeks [2] and led to the hypothesis that early initiating mechanisms should be evaluated at this age i.e. over the weaning period. We have previously established that IAA are present early between 3 and 5 weeks of age and represent a diabetes sub-phenotype since their presence in NOD mouse sera, at this early period, correlated with the final disease phenotype [15]. Thus Early-IAA (E-IAA) have been shown to mark the first measurable phenotypic checkpoint in T1D pathogenesis. Animals positive for E-IAA at 3-5 weeks after weaning, develop autoimmune diabetes earlier (between 16 and 20 weeks) than their E-IAA negative littermates [15]. We have used this E-IAA sub-phenotype to select individual mice as diabetes prone and search for changes in gene expression patterns in the PLN, correlating with the presence of E-IAA in the NOD mouse sera.
Overt T1D is considered to require the dominant presence of islet-destructive pro-inflammatory T lymphocytes [16]. The onset of T cell activation is known to deploy within organized secondary lymphoid tissues, draining the tissue that delivers the adequate signals for activation of T and B lymphocytes.
In the NOD mice, any early islet-disruptive remodelling process taking place, whether it implies β cell apoptosis or repair mechanisms [17], might result in delivery of immune cell-signalling to the PLN. The endpoint of such signalling might include deletion of autoreactive regulatory T cells, and/or expansion of autoreactive pro-inflammatory T lymphocytes as well as T cell-dependent autoreactive B lymphocytes. The later further differentiate as insulin auto-antibody secreting plasma cells [18]. Antigen specific T cells interact with dendritic cells (DC) within intact explanted lymph nodes (LN), and it has been observed that immunological synapse formation and prolonged interactions between these 2 cell types is followed by activation, dissociation and rapid migration of T cells away from the antigenic stimulus area [19]. Even more strikingly, excision of PLNs at 3 weeks of age protects NOD mice against IAA, insulitis and diabetes development, almost completely, but has no effect when performed at 10 weeks of age [20]. Therefore the time frame of 3 to 5 weeks seems to correspond to the initiation of the autoimmune process in the PLN and the related transcriptional profiling can be potentially sort out in this tissue.
We anticipated that at 5 weeks of age, differential gene expression correlated with the presence or absence of E-IAA, in the PLN of NOD mice, could be detected. This timing allowed for prior tests of E-IAA at 3 and 4 weeks. We used microarray analysis to determine gene expression patterns in the PLN. This approach has the potential i) to provide a global profiling of the modulated genes at this first measurable phenotypic checkpoint in T1D pathogenesis, ii) to allow novel hypothesis to be formulated concerning the initiation of the islet-destructive autoimmune process and eventually to further challenge these hypothesis. We identified ectopic high expression of insulin genes in the E-IAA positive PLN. This data confirm the possible cross talk between the pancreas and its adjacent lymph nodes via at least one pancreas specific autoantigen.
Functional annotations of up-regulated transcripts in the PLN of E-IAA positive NOD mice revealed a gene network otherwise found also to operate during inflammatory and tumour processes. Furthermore, we have identified several transcripts coding for extracellular proteins that have to be evaluated as potential disease pathogenesis associated markers and possibly therapeutic targets. This data represent a "freeze frame" of a first identifiable disease check point, associated with the presence of IAA and corresponding to the early steps of the autoimmune process.
Mice were purchased from Taconic farms (NOD/tac). Pregnant females were tested one week before delivery for the presence of IAA. To discard any possibility of trans-placental antibodies affecting our experimental design, based upon the spontaneous appearance of IAA, we have used, when possible, individual mice from progenies of negative females and from litters that they contained both positive and negative individuals.
Animals were tested as previously described [15], at 3, 4 and 5 weeks of age for the presence of IAA. PLN of IAA positive and negative animals were used for RNA preparation and pancreata have been also isolated for histology. All animals were kept under SPF conditions and all studies were performed under the recommended Laboratory Animal Care committee of the University of Colorado.
IAA were measured in a standard radioimmunoassay as previously described [15,21] incorporating competition with unlabeled insulin and precipitation with Protein A/G sepharose in a 96 well filtration plate.
Tissues were formalin (10%) fixed and embedded in paraffin. Sections of paraffin-embedded tissues were stained with Hematoxylin & Eosin and with polyclonal guinea pig anti-insulin antibodies (Millipore) followed by incubation with a peroxidase-labeled anti-guinea pig IgG antibody (Kierkegaard & Perry Laboratories Inc.). The presence of insulitis has been evaluated by visualizing more than 10 islets in 3 different sections from the same individual. Similarly the presence of insulin expressed in the PLNs has been evaluated in more than 3 sections of each individual tissue.
PLN have been dissected from six E-IAA negative and three E-IAA positive animals at 5 weeks of age. Tissues were immediately placed in RNA later containing RNases inhibitors (Qiagen) and processed for total RNA preparation on the same day with Qiagen mini-RNA commercial kits (RNeasy) with on-column DNase treatment following the manufacturer's protocol. Quantity, purity and integrity were confirmed initially by spectrophotometry (A260/A280 ratio) and capillary electrophoresis (2100 Bio analyzer; Agilent Technologies, Palo Alto, CA). 4.5 μg of total RNA were used for the target preparations and no additional step of cDNA amplification has been used in our studies.
Target preparations and chip hybridizations have been performed as recommended by the manufacturer MG_U74A_version 2 GeneChips (Affymetrix, Santa Clara, Ca). Each chip contained 12 486 probe sets (Supplemental Materials and Methods). All gene expression data discussed in this report have been deposited in NCBI's Gene Expression Omnibus [22] and are accessible through GEO Series accession number GSE15582 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15582.
Genes used for hierarchical clustering have been also used to create the Genome view of our transcriptome data by using the dChip software (map of genes tool), as described in the dChip site http://www.dchip.org.
P-values have been calculated for all stretches containing ≤20 selected genes to assess the statistical significance of gene proximity and the significant P-values have been reported in the analysis view. The "tightness" i.e. the rank distance of the genes on the two ends of the stretch was calculated by/n genes against that of n genes randomly put on the chromosome as described http://biosun1.harvard.edu/complab/dchip/map%20chromosome.htm.
Gene Ontology analysis was carried out using DAVID NIAID/NIH online functional annotation tool and ontology terms were retained according to the significance of P-values for any particular GO term. This indicated that there were more genes associated with this GO term than would be expected by random chance [23]. The results were independently confirmed by applying the GO Browser tool in NetAffx to the same set of differentially expressed genes. The associations between altered genes or pathways were further evaluated using the Ingenuity Pathways Analysis software (IngenuityR Systems).
Total RNAs from PLN of E-IAA positive and negative mice were used for Real Time PCR following the ABI software and method. To confirm gene expression levels identified in the arrays by RT-PCR, RNAs were aliquoted from the same samples used for the array hybridizations. Sequences corresponding to the investigated genes have been retrieved from the public database and aligned with Affymetrix target sequences using BLAST software http://blast.ncbi.nlm.nih.gov/Blast.cgi. Primers and TaqMan probes [see Additional File 1] have been designed using the proprietary TaqMan software. We verified that the amplified fragments corresponded to the exact Affymetrix sequences. mRNA from an insulinoma cell line has been used as positive control and 18S rRNA for data normalization.
We have established the following criteria for the selection of individual mice: autoimmune positive animals have been tested at 3, 4 and 5 weeks for the presence of IAA in the serum and selected when positive at least twice [24]. Negative phenotype has been considered when animals were negative for IAA at the same ages. A total of 9 animals have been selected, 6 negative for E-IAA and 3 E-IAA positive (Table (Table1).1). Even though the presence of E-IAA have been demonstrated to be an early marker of autoimmune diabetes, animals negative for IAA throughout their life may develop T1D, yet this remains a rare event (1 animal/15) [25]. In human, some cases of IAA long term positive individuals who do not develop T1D have been observed [26]. As seen on Table Table1,1, regardless of the sub phenotypic attribution to the individual mice used in this study, one E-IAA negative sample (A36.4) was grouped, by clustering analysis, together with the E-IAA positive group, according to gene expression patterns (see below). This observation underlines the complexity of the autoimmune condition and indicates that the balance between pathogenic and physiological immune responses controls the outcome of disease. One possible explanation for the absence of E-IAA in the A36.4 PLN sample is that the selected sub-phenotype marks the disease stage without being per se the cause for the expression patterns identified. Therefore the initiation of the autoimmune process might be dependent upon the identified gene signatures and independent of the presence of IAA in the serum. In this case the E-IAA would be the consequence of an ongoing mechanism but not the cause.
Our interest in this study was to identify gene expression variations solely due to the presence or absence of IAA, in a set of identical animals of the same age and same genetic background, which however are not synchronized for the evolution of the disease. Albeit the NOD strain is inbred, homeostatic differences inherent to each animal might influence the physiopathological status and in consequence the disease stage. Therefore, RNAs have been prepared from PLN, isolated from individual mice and used without prior amplification. This allows identifying and controlling for inherent biological variability of gene expression, between individual animals that might not be related to the E-IAA sub-phenotype and allowed us avoiding the possibility for any preferential transcript amplification.
The quality of whole gene expression data generated from each of the 9 individual animals has been checked using box plots metrics representation [see Additional File 2]. The mean fluorescence intensity values and the range of signal amplitudes were similar across all samples and consistent with a good hybridization quality of the arrays. A scatter plot of the raw data for all transcripts of the arrays from either E-IAA negative or positive samples clearly showed several probe sets to be differentially expressed between the 2 conditions (Figure (Figure1).1). Expression levels for the up-regulated genes were higher in E-IAA positive PLN, while up-regulated variations in the E-IAA negative samples have been observed at a lower level (Figure (Figure11).
Statistically significant differences in gene expression levels between the E-IAA positive and E-IAA negative lymph nodes were determined (adjusted P-values < 0.05 and log2 fold changes >0.42). A total of 177 probe sets were deemed statistically significant by these criteria and corresponded to 165 unique genes, whilst 12 transcripts were identified repeatedly because they were represented on the chip by more than one probe set [see Additional File 3]. Unsupervised hierarchical clustering of the modulated probe sets confirmed clear sample segregation in the two phenotypic groups (Figure (Figure2).2). Gene expression patterns correlated well with phenotypic evaluation for all individual mice except one: sample A36.4 is classified with the positive group (Figure (Figure2,2, noted by *) whilst its phenotypic evaluation, as described above (Table (Table1),1), has identified it as a negative sample. This discrepancy, as discussed above, correlates with previous findings in human and the NOD mouse, whereas autoantibody negative individuals have still a probability to develop the disease [25,27].
Interestingly, 77% (119 genes and 8 ESTs) of the differentially expressed transcripts were up regulated in the E-IAA samples while only 22% (34 genes and 3 ESTs) were down regulated. The majority of the up-regulated transcripts (69%, ie. 82 transcripts and 5 ESTs) have expression patterns varying over 2 fold while only 5 out of the 34 down-regulated genes and one out of the 3 ESTs (16%) showed over 2 fold differences between the two sets of samples [see Additional File 3].
The biological relevance of these trends in gene expression changes will obviously need to be tested on an individual gene basis.
In order to validate the expression levels observed in the arrays, we selected 4 genes for quantitative real time PCR analysis (Q-RT-PCR): 2 regenerating islet-derived coding genes, Reg2 and Reg3a (Figure (Figure3A3A &3B) and the 2 insulin genes, Ins1 and Ins2 (Figure (Figure3C3C &3D) that have shown high gene expression differences in the arrays [see Additional File 3]. Similar trends of gene expression differences between the two sets of samples, in the arrays and in the Real time PCR were observed (Figure 3A-D).
Expression of insulin genes in the PLN was not expected since insulin is synthesized, stored and secreted by the β cells of the pancreatic islets in a highly regulated manner in human and mouse. However ectopic expression or so-called illegitimate transcription of self-antigens has been previously reported, mainly in the thymus [28] and spleen [29]. Cells expressing pancreatic autoantigens are also found in peripheral lymphoid tissues and the same cell can express more than one self antigen [15,30]. The functional significance of these observations has been questioned and evidence from studies in the mouse strongly suggested that self-antigen-expressing cells, in the thymus, may participate in negative selection and mediate tolerogenic signals, some of which may culminate in the apoptotic death of autoreactive lymphocytes [31].
Considering this data, we have stained NOD PLN with anti-insulin antibodies by immunohistochemistry (Figure (Figure3F)3F) and used pancreas as positive control (Figure (Figure3E).3E). Sparse distribution of insulin expressing cells was noted in the PLN (Figure (Figure3F),3F), while no staining has been obtained in inguinal lymph nodes used as negative controls (Figure (Figure3G),3G), thus confirming the ectopic insulin gene expression observed in our data. It is interesting to note that insulin genes have been also found expressed in the PLN of NOD mice at 4 weeks in a recent publication [32] although at lower levels.
Noteworthy that except insulin no other endocrine-specific transcripts (somatostatin, glucagon, somatostatin receptor or Islet amyloidal polypeptide) have been identified in our data set [see Additional File 4]. As expected and similarly to the endocrine specific transcripts, no other transcripts known to be specific either only to the exocrine pancreas or to the developing pancreas were expressed in the PLN at 5 weeks [see Additional File 4]. While expression of insulin genes in the PLN is not a novel finding, the high levels of expression found in the PLN of E-IAA positive NOD mice was not expected and is bewildering. One explanation might be that in the E-IAA positive PLN, APCs expressing insulin might act, locally in the PLN, as insulin-presenting cells. This would contribute to lack of tolerance to this molecule and consequently to the instigation of insulin autoreactive lymphocyte trafficking, leading to infiltration of the islets resulting to T1D. However a role of insulin in local tissue architecture, independent of its autoantigen function, cannot be excluded.
An important aspect of our experimental design concerns the early selection of NOD mice by the presence of E-IAA, used as a marker of early autoimmune activity, at an age whereas homeostatic changes in the pancreas might take place. It has been demonstrated that increased neonatal beta cell apoptosis is taking place with a peak at 2-3 weeks after birth, in T1D animal models including the BB rat and the NOD mouse [33]. The cross-talk between the pancreas and the adjacent LN during or immediately after this phase might be reflected by discrete gene expression changes in this later tissue.
We undertook a series of exploratory functional data analysis aiming to comprehend the roles of the identified gene expression variations in E-IAA-dependant islet autoimmunity. Forty-one out of the 119 up-regulated genes showed differential expressions over 5 fold [Additional File 3]. This indicates that the autoimmune process as reflected by the presence of IAA in the positive NOD mice is marked by local gene expression changes in the PLN. This might include changes in the immune process, occurring in this lymphoid tissue and/or changes in local tissue architecture and remodelling, not only of the lymph node but also of the pancreas, as mentioned above, assisted or mediated by the genes expressed in the PLN.
An over representation of genes coding for extracellular proteins has been identified as 38% (62/165) modulated genes belonged to this category (P-value = 8.49E-16) (Table (Table2).2). This does not seem to be a random distribution since the first array of the MG-UG74Av2 series contains 2,057 transcripts coding for secreted proteins in a total of 12,488, representing 16% of the total number of genes on the arrays. Moreover this class of proteins is overrepresented in the E-IAA positive list of genes in the PLN (Figure (Figure4a).4a). The majority (30/41) of the over 5 fold expressed genes in the up-regulated samples, code for secreted proteins (GO P-value = 3.46E-22). This might reflect the cross talk taking place between the PLN and the pancreas, during the pre-inflammatory stages. Moreover secreted and membrane-associated proteins are an important class of proteins, as they have the potential to be detected in biological fluids and thus may be potential diagnostic markers or therapeutic targets.
Genes coding for secreted proteins include the Spp1 gene coding for osteopontin that has been found to play also a role in autoimmune demyelinating disease (MS) in human [34]. High levels of this pro-inflammatory cytokine have been reported in active relapsing-remitting MS patients [35]. Several other genes coding for secreted proteins with an immune/inflammatory function, include kallikreins genes (Klk1b22, Klk1b9, Klk1b5, KlK1rb9, Klk1b1, Klk16) that have been described as potential biomarkers for certain cancers [36]. Members of the kallikrein large family that belongs to serine-type proteases have been implicated in autoimmune diseases, in particular in a model of Sjögren's syndrome [37].
In contrast to the high representation of secreted proteins coding genes, nuclear proteins transcripts are more rare in our data set, representing 11% of the total number of the modulated genes (18/165) (Figure (Figure4a).4a). This could indicate that the high levels of mRNA found in the E-IAA positive PLN reflect a natural steady state process of gene transcription, initiated prior to 5 weeks and/or regulation of the mRNAs is taking place at the post transcriptional level.
Aiming to correlate relevant functions of our transcriptome data with the E-IAA sub-phenotype and the end T1D phenotype, we assessed for functional significance of the identified genes. We applied two different approaches: first the identified differentially expressed genes between E-IAA positive and negative NOD mice have been subjected to Gene Ontology analysis (GO, EASE) [23] and evaluated for classification according to functional annotations for over-representation, in combination with assessment by PANTHER terms resource [38]. Thus in each category are included not only genes belonging to known functional categories by GO terms but also genes for which annotations are attributed by existing published experimental evidence together with consideration of evolutionary relationships (PANTHER).
In this analysis, terms with the highest levels of significance have been retained (for GO 8.35E-5<p < 0.07 and for PANTHER 1.2E-4<p < 0.02). Several genes have been annotated to Immune related process (Table (Table2)2) and several belong to the serine proteases group and include inflammation and immune functions (Table (Table2)2) and [see Additional File 5]. This group of proteins is characterized by the presence of a serine in the catalytic domain that is highly conserved between its members. Disorders of the dynamic balance of proteases and their inhibitors have been reported in the blood stream and in lymphocytes of diabetes patients and it has been proposed that increase of proteases concentrations represents an early marker of T1D [39]. Imbalance of the proteolytic system might also change the local homeostasis and interfere with the extracellular matrix of cells in the pancreas that become more permissive to lymphocyte invasion as it has been reported for tumour metastasis [40]. Further experimental evidence is required in order to comprehend the significance of the presence of serine proteases in the PLN of NOD mice.
Concerning the relevant distribution of functions between up- and down regulated gene lists, it is to be noted the presence of the serine-type proteases coding transcripts, found only in the up-regulated list (Figure (Figure4b).4b). In contrast, 5 antigen binding genes were all down-regulated in the E-IAA positive animals. Several immunoglobulin genes, including genes coding for the constant region of the kappa chains and the lambda variable region 1 are in this category as well as the Cd1d1 antigen (Table (Table3).3). Cd1d1 plays a critical role in the positive selection of T cells that produce Il-4 [41]. It has been reported that CD-1 reactive NKT cells are required for the development of systemic tolerance and defects of NKT cells affect autoimmunity [42,43]. NKT cells while they enhance the response to some bacterial, viral and parasitic infections and they play a similar role in some types of cancer, yet can suppress autoimmune disease, allograft rejection and graft-versus-host disease [44]. Therefore up-regulation of the Cd1d1 molecules in the E-IAA negative lymph nodes might prevent the development of autoimmunity via its binding to NKT-TCR. This can lead to expression of Il-4 by NKT cells [45]. Il-4 is known as a Th2 cytokine and therefore protective for T1D [2]. A similar protective mechanism has been described for experimental autoimmune encephalomyelitis (EAE), considered to be the prototype autoimmune disease mediated by type 1 helper T (Th1) cells, in the mouse [45].
In a second approach we used literature mining together with Ingenuity functional annotation tool (Ingenuity® Systems). Combination of the resulting functional annotations for all the genes in the list [see Additional File 6] allowed assignment of more than one biological function to certain genes and gave an overall classification in twelve main functional categories (Table (Table3).3). Genes that did not clearly belong to any of the categories were classified together with ESTs, as 'unknown' and comprised less than 12% of the modulated genes (Table (Table3).3). In our data set ESTs represented 7% of the identified genes, while in the chips, unknown expressed sequences represent approximately 50% of the 12,486 probe sets spotted (MG_U74A_v2 arrays).
Several transcripts belonged to more than one category (Table (Table33 and Additional File 6). Given the essential role attributed to the PLN in the activation of islet-specific immune response [46], it was assumed, at this pre- or peri-inflammatory stage (5 weeks of age) in the NOD mice, that several chemokines or adhesion molecules would be modulated in the autoantibody positive mice. However this has not been the case. While several inflammatory mediators were consistently expressed in all PLN from E-IAA positive and negative mice, few of these molecules were expressed in a significantly regulated manner (data not shown). This concerned genes coding for several chemokines (CCR7, CXCl12, CCR5, CXCl13, CCl21a and CXCl9) that although gave present calls (P) to all samples, did not meet statistical criteria for significant differences between the two groups (data not shown). One hypothesis might be that although all NOD mice possess the necessary requirements (i.e. presence of several chemokines in all samples) for the initiation of the inflammatory process against the pancreatic islets, the autoimmune-driven destructive events are probably dependent upon the levels of pancreatic tissue remodelling taking place. This in turn could be due to either antibody bearing B cells (IAA) or T lymphocyte specific trafficking against pancreatic signals.
A genome view of our transcriptome data gave an unexpected image of chromosomal gene localization, pointing to mainly two chromosomes that showed stretches with the highest P-values containing >20 selected genes (Figure (Figure5).5). This was the case for chromosome 6 (27 probe sets, p = 1.31E-5) and chromosome 7 (23 probe sets, p = 0.025). Two additional stretches on chromosome 10 and on chromosome 17 around the MHC locus were also detected but with a lower gene number.
Genes on the chromosome 6 are placed in 3 families including the lithostatin family, immunoglobulin light chain and several serine type proteases coding genes. Genes on chromosome 7 concerned mainly the kallikrein coding genes and the insulin-2 gene. Interestingly 4 T1D Idd loci have been described on these two chromosomes by genetic linkage analysis: two loci on mouse chromosome 6, the Idd6 [47] and Idd19 [48] and two Idd loci on mouse chromosome 7: the Idd7 [49] and the Idd27 [50].
Autoimmune diabetes in human is a heterogeneous disorder whereas several genes influence the apparent clinical phenotype together with environmental factors and homeostatic differences inherent to the organism. This leads to phenotypic variations of the disease observed not only in human but also in the NOD mouse, despite the genetic homogeneity of this inbred strain. Indeed autoantibody responses vary between individual mice by the titers and by the age of appearance. Similarly temporal variation in the severity of the islet-damaging inflammatory process is observed as well as variations in the onset of appearance of the end point disease.
In the present study we used the early presence of IAA in the NOD serum, as the earliest measurable sub-phenotype, aiming first to address for changes occurring in the pancreas-draining lymph nodes, when the autoimmune process has been just initiated. The PLN is a structured secondary lymphoid organ where pancreas derived cells and signals are expected to be delivered. The resulting output of the processing of these signals is either the maintenance or the loss of pancreas integrity. Our second aim was to attempt a stratification of disease check points by studying the first possibly identifiable disease stage, as selected by the presence of E-IAA.
This rational, potentially, allows identification of early genetic components that due to their specific expression in the PLN, at a precise time, may not be identified by genetic studies whereas the final T1D phenotype is usually taken in consideration.
Phenotypic stratification, as delineated by sub phenotypic attribution for the identification of gene expression variation in autoimmune diabetes, has not been used, to our knowledge, prior to our study. Two recent reports have also used PLN in a similar methodology approach, at early ages in the NOD mice, without however prior phenotypic evaluation [32,51]. One group studied 4 and 6 weeks old NOD/BDC 2.5 mice [51], the other group used NOD mice of various ages starting at 10 days and including 4 weeks, to compare with NOD.B10H2b mice [32].
While our study has been carried out at 5 weeks of age it differs from these former investigations by the experimental design, based on the E-IAA sub phenotypic animal selection criteria. Accordingly, it can be assumed that gene expression changes solely correspond to the phenotype used for selection since the only perturbation, in our design, is the presence of E-IAA. While we cannot exclude that the identified gene profiles might correspond to the more rapid, though still infra-clinical progression to T1D, of the individual mice rather than to the E-IAA sub-phenotype, hierarchical unsupervised clustering (Figure (Figure2)2) indicates however that samples are not randomly distributed. As the genetic background and the ages of the animals are indeed identical, this gives the opportunity to identify genes that might be activated in an autoimmune-dependent manner in the NOD mice, after or around the massive neonatal β cell apoptosis taking place [33]. It has been suggested that β cell death throughout the early ages in the NOD mice, may be an initial trigger for APC activation and the development of T1D [52]. Defects in silent clearance of apoptotic remnants before necrosis might prevent processes of the physiological maintenance of tissue integrity to operate [53]. In the rat intestine a discrete subpopulation of DCs has been found to transport apoptotic remnants to T cell areas of the mesenteric LN [54]. Interestingly several transcripts expected to contribute to physiological tissue remodelling were up-regulated in the E-IAA positive list (Table (Table33 & Figure Figure66).
The expression of five genes belonging to the regenerating islet-derived gene family (Pap, Reg1, Reg3a, Reg3g and Reg2), implicated in tissue integrity, maintenance and regeneration were highly up-regulated in our data set [see Additional File 6]. They code for a set of related but distinct proteins [55]. The presence of these proteins in the E-IAA samples is intriguing, especially in the light of recent published reports demonstrating that adult human islets possess a remarkable degree of morphogenetic plasticity [56]. Similarly to Reg genes, Itih-4, a serine-type protease inhibitor with anti-apoptotic and matrix stabilizing functions, is also up-regulated and reported to play a role in liver development and regeneration [57]. These transcriptional signatures, if sorted out as such, might represent a new tool in the understanding of islet neogenesis and tissue preservation by inherent factors and could be valuable against β-cell destruction.
Several of the modulated transcripts belong to complex networks of gene products acting in tissues during inflammatory sustained processes (ie. Spp1, Klks, Mt2), or playing a role in tissue regeneration (ie. Regs) and have been found also to be associated with neoplasia (Table (Table3),3), [see Additional File 6]. Certain kallikrein coding genes have been up-regulated in our data set and are considered possible biomarkers for cancer [58]. Similar expression patterns have been found for Muc1 gene that is highly expressed in gastric carcinoma and proposed to be involved in gastric carcinogenesis [59] as well as for the genes Cckar, Ggh [60,61] and Ang [62] that were reported to be implicated in the genesis of various tumours. Ang, in particular, is involved in the Notch signalling pathway related to neurovascular progression of pancreatic cancer and metastasis [63], as well as in multiple myeloma [64]. It has been reported that circulating levels of angiogenic cytokines can predict tumour progression and prognosis in neuroendocrine carcinomas of the gastro-entero-pancreatic system [62].
Finally, the gene coding for nucleobindin 2 (Nucb2) implicated in B cell lymphomas [65] was also up-regulated. It is a DNA binding protein, called also NEFA and its sequence contains a signal peptide, suggesting that it is a secreted or trans-membrane protein [66].
Several genes known to play a role in various inflammatory conditions were differentially modulated in our data set (Table (Table3).3). Such molecules include two elastase genes, Ela1 [67] and the neutrophil elastase Ela2 [68], known to play a role in peripheral tissues invaded by microbes [69], the Spp1 gene [70], and the secretory granule protein syncollin (Sync), expressed by neutrophils with a role in host defence to infections by invading bacteria, fungi, and protozoa [71]. Sync is also expressed in the exocrine pancreas and in the duodenum and colon [72]. Other gene products operating during the inflammatory process include the Egfbp2, part of the kallikrein gene family [73], the actin binding protein (Actb), the mentioned above Muc1 that is part of signalling proteins regulating cell adhesion/de-adhesion [74] and two genes coding for metallothionein, Mt1 and Mt2. Increased levels of Muc1 have been found in the small intestine of a cystic fibrosis mouse model [75]. Over expression of metallothioneins has been described in gastric cancer and in intestinal metaplasia and dysplasia [76]. Mice lacking metalothionein are more susceptible to Helicobacter pylori colonization and gastric inflammation, indicating a protective effect of these proteins against H. pylori-induced gastritis [77].
Interestingly, several lipases coding genes that have been associated with diabetes were up-regulated in the E-IAA positive PLN (Table (Table3).3). Pla2g1b gene has been described as a pro inflammatory molecule expressed in lung epithelium hosting Pseudomonas aeruginosa [78]. It is also implicated in glucose uptake by the liver, heart and muscle tissues under high glucose diet [79]. Inhibition of lipase activity was shown to reduce the incidence of type 2 diabetes mellitus and genetic variations in the Clps and Pnlip genes are associated with type 2 diabetes [80].
Two additional genes coding for pancreatic lipase related proteins Pnliprp1 and Pnliprp2 were highly up-regulated in the E-IAA PLN (Table (Table3)3) and [see Additional File 3]. Pnliprp2 protein plays a crucial role in the digestion of dietary fats in milk suckling mammals [81] while it seems to participate in T cell cytotoxicity. A T cell lipase shares 74% amino acid identity with the pancreatic Pnliprp2 [82].
Overall the data reported herein suggest that the events leading to islet-damaging autoimmune destruction might be rooted to early processes, some sharing common characteristics with several other inflammatory conditions found also in microbes-hosting tissues (i.e. Pla2g1b, Mt1) and in certain cancers (i.e. Klk genes, Muc1, Reg genes). Noticeably among the cytokine receptor-like factor 1 and 3 (Crlf1, Crlf3) that belong to the same family [83], Crlf3 has been found to be up-regulated in skin cancer [84]. While Crlf1 expression is up-regulated in the E-IAA positive samples, expression of the Crlf3 gene is down-regulated. Crlf1 mRNA has been reported to be up-regulated by TNF-α, IL-6, and IFN-γ [83]. Polymorphisms in the regulatory region of C1qb gene have been correlated with down-regulation of the murin c1q protein levels and linked to lupus nephritis [85].
The exact order of events in autoimmune initiation requires additional studies in order to be elucidated. Our hypothesis implies that defects in pancreas remodelling processes, taking place at the weaning period, might be a trigger for immune deregulation leading to autoimmune tissue destruction. This might include tissue regeneration or damage. The developmental stage of the organism as well as agents exogenous to the organism, such as invasive microbes, potentially can contribute to local homeostatic changes. It can be assumed that the identified genes have the potential to re-address the question of the link between environmental triggers, such as sustained microbial signalling processes, with the autoimmune process, at least in T1D.
This hypothesis requires certainly additional integrative studies and experimental evidence to be validated. It is noticeable that none of the common pro-inflammatory molecules such as tumour necrosis factor or interferons have been found in our group of genes. It has been recently reported 8 IFN-α inducible genes to be differentially up-regulated in CD4+ T cells isolated from the PLN of 6 wks, when compared with 2 weeks old NOD/BDC 2.5 mice [51]. These authors concluded that IFN-α initiates T1D in the NOD mice and suggested that pDCs and TLRs play a role in T1D initiation. Though NOD mice differ from strains unable to initiate islet autoimmunity in terms of their response to IFN, our data indicate that such differences are not associated, within the NOD strain, with early expression of IAA, which in its turn is associated with greater risk of diabetes progression, at least at the age studied in this report.
The autoimmune-related transcriptional early landscape of the PLN, in a first glance, seems difficult to be contained due to its complexity. The stepwise nature of the process resulting in T1D, is illustrated in our study by the difficulty to sort out a clear mechanism describing a possible scenario for disease initiation and implicating, preferably, a small number of molecules. Nevertheless, the gene signatures identified by this global analysis of the PLN in the autoantibody positive NOD mice at 5 weeks, together with the functional annotations described herein, represent a valuable tool in designing additional experiments for understanding the irreversible initiation of the autoimmune process in T1D. Experimental investigation of the functions of the identified genes together with further integrative analysis studies have the potential to sear light into the exact autoimmune-related processes that take place at this early age, in autoimmune prone, insulin autoantibody positive animals.
T1D: type 1 diabetes; NOD: Non Obese Diabetic; PLN: pancreatic lymph node; DC: Dendritic Cell; APC: Antigen Presenting Cells; BCG: Bacillus Calmette-Guérin; Idd: insulin-dependent diabetes locus.
All authors are free of any commercial affiliation or consultancy or other arrangement that could be considered to pose a conflict of interest regarding this submitted article.
EM and DM performed experiments, BR and EM analyzed data, JOyF participated in functional annotations analysis; EM conceived and coordinated the study, EM and GE designed the study, EM wrote the manuscript, GE supported the experimental part of the study in his laboratory.
All authors read and approved the final version of this manuscript.
The pre-publication history for this paper can be accessed here:
Sequences corresponding to probes used as primers for amplification of selected genes by Real Time Q-RT-PCR.
Quality evaluation of the arrays by box plots metrics. RMA-normalized data distribution for the E-IAA positive and the E-IAA negative groups
Genes differentially expressed in the PLN of 5 weeks old NOD mice according to E-IAA sub-phenotype and adjusted P-values.
Genes specifically expressed in the pancreas but not found to be expressed in the E-IAA PLN transcriptome.
Functional categories of genes and ESTs as annotated by GO and PANTHER tools. Molecular functions and Biological process terms are as described in the text for Table Table22.
Assignment of functional categories as annotated by literature data mining, for genes and ESTs differentially expressed in the PLN of E-IAA NOD sub-phenotype, as described in the text (Table (Table33). References, corresponding to bibliographic search for functions for each gene, are given.
We thank Rebecca Brown for assistance to initial analysis of the data and Geneviève Milon and Eric Prina for critical reading of the manuscript. EM is endeavoured to Genevieve Milon for support and encouraging discussions. This work has been supported by the BDC, University of Colorado and the Institut Pasteur, Paris.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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In a recent promotion, Waunakee Community Bank donated $50 to a local charity whenever someone signed up for a new account. Eleven people picked WNC as their charity of choice, and we are so honored! Thank you to Waunakee Community Bank for supporting local organizations, and thank you to those 11 individuals for choosing WNC!
We look forward to working with WCB as we continue our partnership to Build a Stronger Community!


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So how do you enter? It's easy. Just a) leave a comment telling me your favorite Halloween costume and if any of it will go into your photobooth. And b) leave your email. And c) you have to tell me you promise to pass along the camera for free, with whatever film you have left, when your wedding is done. Because that's how wedding magic grows. And this camera is all about building and capturing wedding magic.
Friday, June 10, 2011
Fuiji Instax Mini Giveaway!
So how do you enter? It's easy. Just a) leave a comment telling me your favorite Halloween costume and if any of it will go into your photobooth. And b) leave your email. And c) you have to tell me you promise to pass along the camera for free, with whatever film you have left, when your wedding is done. Because that's how wedding magic grows. And this camera is all about building and capturing wedding magic.
21 comments:
My favorite Halloween costume was probably this year's statue of liberty circa 1890, that is, before the green patina set and it was still copper colored. I will probably put the torch and crown in the photobooth prob box, but the fabric I used for the robe has been repurposed for our chuppah, so that is unavailable. My email is firstname.lastname@example.com
Thanks for paying it forward with this rad giveaway!
I've loved all of my costumes, but I'm really hoping this year we can pull off Inspector Gadget and Penny. If we do, THAT will be my favorite.ReplyDelete
We want to have a wedding booth, but with all of the other detail garbage that's taken over our lives, we haven't gotten around to actually organizing it yet. It's looked like it's going to fall by the wayside, which would be a shame. I would mos' def pass this ish along to future brides. Probably also in cute packaging. Just saying.
My email address is email@example.com.
My favorite Halloween costume was Wilma from the Flintstones. I threw it together last minute from a thrift store find (leopard print skintight dress, teased hair) that probably came straight from the trailer park. Unfortunately, it died that night from too much good times. The photo guest book booth props are going to come mostly from a care package my sister put together that includes sunglasses big enough to wear as a bra. Email is firstname.lastname@example.com
You and Maggie are both RAD, I feel so lucky to have stumbled into this community! I've already gotten so much love I'll definitely pass it along.
- Nicole MJune 10, 2011 at 6:53 AM
My favorite Halloween costume was my Hot Librarian year (not what it sounds like). I worked at a rare book library and a month before we had had a fire. No books damaged but still scary. So for Halloween I took one of my work polos w the library logo on it and ripped it up and literally burned it with a lighter. It was singed and a total mess. Paired with a miniskirt, sexy librarian glasses and burnt-cork smudges all over my face and arms and I was a Hot Librarian (get it???).ReplyDelete
I like it bc it was clever, but the reason it is my favorite is that party was the first time my fiance (a former coworker) and I really socialized and, dare I say, flirted.
The glasses will go in the propbox along w the clown noses from our engagement photos and all the other crazy we have in the house. We are theatre folk.
Thank you so much for this community and this blog. The sanity that emanates from here has gotten me through more wedding obstacles than I can count. email@example.com
haha. My favorite halloween costume was when my fiancé dressed up as a PC instead of a Mac. It was great and imaginative.ReplyDelete
I'm not sure my fiancé and I will have a photo booth, but we are collecting a variety of cameras to be used at our reception. We are not having a photographer, so a Fuji Instamax would be awesome to add some variety to the images are guests shoot.
I have had so much help through craigslist and buying used items from other brides that I would love the opportunity to pass the camera along to another bride-to-be. My e-mail is aandj8804[at]gmail.com Thank you for passing on something so fun!
Definitely my Michael Myers (from Halloween) costume. Under the mask no one can tell who you are, and I ran around with a big bloody knife all night. Fun times. The knife may make it to the photo booth, but I don't think many people will want to mess up their hair with the mask....ReplyDelete
And yes, I promise to pass! I'll even come back here to pass it on if you like.
My favorite halloween costume is one yet to be put into action - I really want to dress up my dog as an ewok! I know,it would make me one of "those people" who dresses up her dog but he looks like an ewok anyway and is adorable.ReplyDelete
I don't think I have the energy to create a photo booth, but I would love it next to our guestbook. We plan to do a photo line (photos of us on a string with clothes hangers) and it would be nice to have people add their own pics to the line!
And of course I'd pass it along, in fact, I'd give it back to you to pass along since you probably have a bigger community.
- AnonymousJune 10, 2011 at 12:01 PM
Your post just made me have an ah-ha moment! I'm cliping escort cards on ribbons and was trying to figure out how to to use that for our guestbook as well - and it would be great to have everyone replace their escort cards with a silly/fun photo and the size would be PERFECT. Really though.ReplyDelete
And it actually might get used at our rehearsal lunch/picnic too.
I hope who ever wins uses it with joy!
eabshere at gmail dot com.
Oops, I didn't leave my email:ReplyDelete
mrk41709 at gmail dot com.
I didn't know about this! This is so damn sweet it's got me all teared up. You keep making me cry happy, sappy, wedding tears!ReplyDelete
I can't pass this along, but my fave Halloween costume was my first Halloween with The Candyman. I took 70's prom outfits and ran over them a few times with the car. I rubbed dirt on them. Then I did zombie makeup on us both and we were the Dead Zombie Prom Couple. The Candyman looked a lot like Beetlejuice.
My favorite costume was the Daffy Duck costume I made in Junior High school. That thing survived for 15'years, won costume contests and was responsible for busting high schoolers who were caught smoking behind the school when my mom wore it one Halloween. Alas, it finally bit the dust last year.ReplyDelete
I was thinking it might be a blast to create a HS Prom photobooth for the reception, with a cute blow up of our old prom picture. I of course would pass it along to brides in the blogging community, who have saved my bridal bacon and sanity many times over FHA past year-plus.
Okay, so this post is making me feel all kinds of warm and fuzzy. I can't believe you actually used the box! And posted pictures from both weddings - that's fantastic! I felt sort of bad after our wedding because I'd panicked at the last minute and bought WAY too much film and felt like it was a wasteful "oops" ... but this makes it all feel worth it. :) :) I hope the wedding magic continues for the next couple! (the photos and camera really are a lot of fun).ReplyDelete
And just for kicks: my favorite Halloween costume was "Sexy Lunch Lady," complete w/a hairnet, yellow rubber gloves and a ladle.
My favourite Halloween costume was as a mexican - a poncho, a moustache and a sombrero (:ReplyDelete
I definitely will pass on the camera which has captured so much joy to allow someone else to capture to also capture the wonderful memories.
Thank you for this opportunity (:
My favorite Halloween costume - by far - was MINNIE MOUSE :) I had big sequined ears with a big pink bow, and big pink bows on my shoes, and a big pink polka dotted dress. Of course, I dressed up my black puppy as Mickey and we ended up winning a costume contest which got us a year free at a local puppy dog park, which ended up being the BEST thing ever!ReplyDelete
jessiterrell at gmail . com
Of course I will pass it on! My wedding is in 4 months in October and will be straight insanity.
This is awesome! I don't need to enter cos I have an old polaroid camera I was going to use... and I live in Australia, land of the crazy postage costs. But I think it's such a cool idea and will excitedly follow the camera's progress!ReplyDelete
My favourite halloween costume was actually J's. He went as Clark Kent and had a rippable front white shirt with a superman chest underneath.
Wow! I read this post and just squealed with delight! First of all---I have to say I love your "pay it forward" method :) It's so sweet!!!! And I think your "stipulations" for entering the contest are fun :)ReplyDelete
a) Favorite Halloween Costume would be when the fiance and I dressed up as members from DEVO in 2010. (Pics here: http://bornandraisedinwesternpa.blogspot.com/2010/10/when-problem-comes-along-you-must-whip.html) Those red hats have ALREADY been placed in the "guest book photos box" we have sitting in our living room for the wedding in 34 days! Our friends have been dropping off various props the past month to use for our DIY photoboth.
b. My email is firstname.lastname@example.com
c. I PROMISE with all of my heart that I would pass this on to the next couple. I just have to share that I cannot believe you posted this contest. My fiance and I had our heart set on a photo booth for our wedding--but it just wasn't in the budget. My sister has been trying to think of alternatives for many months. Over memorial day weekend we discovered the Fuji Max camera and came up with a plan to use it to pull off what I envisioned as a cool guestbook/ photo slbum. Unfortunately, I also JUST lost my job (my teaching position was furloughed for next year) so have been debating whether this was something we should financially spring for.
We enlisted a friend to take pictures for us--and we were just going to do "digital" photos that we would develop later and put into the guestbook/photobook that the guests would sign during dinner. However--it would be MEGA cool if we could have them sign AND put the picture in on the same day!!! The Fujimax could make this happen!!!!!
Regardless if we win or not---I really like your idea and thank you for opening this up to your blog readers :)
My bf, I mean fiance (we got engaged yesterday), like to do couples costumes for Halloween. My favorite so far is from 2 years ago. I dressed up as Little Red Riding Hood and he was the Big Bad Wolf. I think it would be hilarious to have the wolf mask and & hairy wolf gloves as part of the photo booth accessories.
I believe in wedding karma and will definitely pass it along to another bride. My email is: email@example.com
Great idea! I'll be honest, I'm not the bride...but I AM the bridesmaid, so hopefully my entry can still count b/c I KNOW that my friend wants one of these for the photobooth at her wedding. I will definitely make sure it gets passed on after her wedding b/c that is a great idea & I will definitely make sure my husband's giant '70s 'fro Halloween wig is in the box of picture props! Thank you! (firstname.lastname@example.com)ReplyDelete
Yay!! My fiance and I are getting married next year in Joshua Tree National Park. We are going completely DIY and definitely want a fun photo booth for our guest book! I saw that they have new polaroid cameras but they are expensive and UGLY! So this would be so rad! My favorite Halloween costume was two years ago when I was Jem (from the early 90s cartoon!) The props at our wedding will likely be fake mustaches and fun vintage picture frames. We want to have a corkboard at the site so people can sign the photos and tack them up immediately. And if I happened to win this awesome camera I would send it back to you so that you could pass it on to someone else who loves your blog!!!ReplyDelete
email: firstname.lastname@example.com
I have been planning to buy a Fuji Instax at our wedding, but was having some insecurity about how it will be slipshod compared to awesome smilebooth-type setups. And then I went to a wedding this weekend, with the fancy photobooth. And I love our silly photos so much, and the fancy red velvet booth and paid person managing the business was so, so unnecessary. I am so excited for all the silly photo memories that will be created at our wedding. My favorite H-costume was when my fiance and I dressed as caterers, from the show Party Down, with plastic champagne flutes velcro-ed to a platter. Everyone thought we were actual party staff that were highly incompetent! firstname.lastname@example.com. Thanks so much.ReplyDelete
Love this!! I'm getting married in September and this would make such an awesome addition to our in-the-works photobooth! My favorite Halloween costume was when I dressed as Smurfette, complete with blue tights and blue body paint covering my face and arms.ReplyDelete
Unfortunatly, I think that blue body paint may be a little messy for the wedding (the paint removal process was long and tedious), so the Smurf costume we'll have to skip. We will be filling in where Smurfette left off with a Carmen Miranda fruit hat, Army flight suits, a red feather boa, and an authentic Mexican sombrero, among many others I'm sure.
I would love to be part of this chain of camera good will, and of course will pass it along to another kindred spirit after my wedding. As a side note, I have a cousin who is getting married in July, and I would love to surprise her with some awesome pics from this camera before using it at my own wedding!
Thanks for the chance, what a great idea!
-Marisa (email@example.com)
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CaMarron Dupree H Model
Chase LeGarconModel Male Atlanta, Georgia, US
My MM URL: http://www.modelmayhem.com/GT611
Mayhem # 1371236
About MeHello Model Mayhemers!!! My goal from joining this site is to eventually fufill my life long dream of becoming a high fashion/fitness model. I always wanted to model as early as high school. I've done a few small jobs and signed to a few small agencies and they all were great learning experiences but now I believe i'm ready for the next level. I hope to meet great photograpers and fellow models and I'm sure this is the place to be. I"m open to TFP shoots also, I believe that experience is one of the best tool one has. If interesed my email address is firstname.lastname@example.com Thanks Everyone and Best of Luck to everyone!!!
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View Single Post
April 20th, 2009, 07:34 PM
Join Date: August 2, 2008
Location: Illinois
Re: *** Post ALL Masturbation Discussion HERE ***
Originally Posted by
If it gets
is it still seminal fluid with no sperm? the first time, it was
but every day since then it has gotten thinner (not much color difference, though).
Now that you mention it you raise a good point. My first time was really thick, but subsequent times were thinner. From my experience when one does not masturbate in a while or if one is really, really aroused, then one's semen can get pretty thick. My guess is that if thick semen is truly important to you, then masturbate as few times as you can so that you can get a maximum amount of arousal from your masturbation session.
Personally, as long as the orgasm is good, then it really should not matter how much semen one is ejaculating or whether it is white or clear. Unless one is hoping to conceive a child at this point one's semen consistency really should not matter.


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Cube Steak Diane
Be the first to review
READY IN: 15mins
Recipe by mailbelle
Cube steaks with an interesting combination of ingredients for the sauce: white grape juice, Worcestershire sauce, and Dijon mustard. You can have this from stovetop to table in under 20 minutes! From TOH.
See Discussion:
Ingredients Nutrition
- 4 beef cube steaks
- 1⁄4 cup butter
- 1⁄2 cup white grape juice
- 2 tablespoons Worcestershire sauce
- 4 teaspoons Dijon mustard
- salt and pepper
Directions See How It's Made
- In a large skillet, saute steaks in butter for 2 minutes on each side or until meat reaches desired doneness.
- Combine the grape juice, Worcestershire sauce and mustard; stir into cooking juices. Bring to a boil; cook for 1 minute. Remove steaks and keep warm.
- Cook the sauce 1-2 minutes longer or until it reaches desired thickness. Season with salt and pepper. Serve sauce over the cube steaks.


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Woman Gives Up Career to Help AIDS Patients
By SHARON L. BASS
Published: June 11, 1989
SOME people do not understand why Maureen Cassidy of Fairfield gave up a career as a cosmetics executive, and her paycheck, to work with AIDS patients.
Each day, her new unpaid job takes her to a donated office in the heart of Bridgeport, where rows of orange vans wait in the parking lot to deliver food to the needy.
Ms. Cassidy, who is 38 years old, said her life has changed since last year when she started the Buddy Program, a service that matches volunteers with AIDS patients. The Buddy Program is run out of the Family Services-Woodfield Agency and is part of the Greater Bridgeport AIDS Support Services.
''I walked into a clothing store recently and a woman inside said she couldn't believe I worked with people with AIDS,'' Ms. Cassidy said. ''She said, 'Aren't you scared of getting AIDS?' '' Ms. Cassidy said she uses each confrontation as an opportunity to educate people about AIDS. Deaths of Relatives
Her involvement stems from the AIDS deaths of friends and relatives, including a brother and from the benefits of her work.
''In this world where we hear about how awful everyone is, I see only the good,'' she said, of volunteers and the bravery of the people they serve.
Last month, Ms. Cassidy received a $10,000 grant from the Stewart B. McKinney Foundation in Fairfield to cover operating expenses and start-up costs, like brochures and business cards. This is the first grant the program has received, she said.
The Buddy Program is modeled after similar ones at the Gay Men's Health Crisis Center in New York City and the Shanti Project in San Francisco. Ms. Cassidy had volunteered with the New York group for six years. One of a Kind
She said the Buddy Program was the only one of its kind in its service area, which includes Bridgeport, Easton, Fairfield, Monroe, Stratford and Trumbull. The program has about 100 volunteers who work with AIDS patients, prepare meals for them or perform clerical tasks.
Volunteers include the elderly, business professionals, doctors, nurses and homemakers - ''everybody and anybody,'' Ms. Cassidy said. ''They are just kind, good people who realize AIDS is a national crisis and they want to do something about it.''
According to the Bridgeport Health Department, 4,000 residents are infected with the AIDS virus and 103 are ill with the disease. In the surrounding towns that are served by the Buddy Program, 200 people are infected. Figures are not kept on those with AIDS. The numbers are probably higher, Ms. Cassidy said, because some people who suspect they may be infected go elsewhere for testing.
Buddy volunteers go through a 20-hour training program, run by Ms. Cassidy, to learn about the basics of AIDS, death and dying. At the workshops, people with AIDS and health professionals speak to the group. Volunteers are then required to put in 5 to 10 hours a week. Two volunteers are usually assigned to a client to help with errands, cooking, cleaning and doctors visits, and, most of all, to offer companionship.
''They listen to them when nobody else will,'' Ms. Cassidy said. 'A Great Sense of Humor'
A client with whom Gary Larson had been working with died recently. He was a 30-year-old disk jockey who worked in New York City. ''He was suicidal, very lonely and watched TV all day,'' said Mr. Larson, a Fairfield resident who is a retired college professor. ''All he could eat was milkshakes. But he had a great sense of humor.''
Mr. Larson said they spent much of their time laughing and talking. The final days were ''very painful to see,'' Mr. Larson said. Volunteers often play advocacy roles when AIDS patients are admitted to hospitals. ''A lot of times they have to go in and fight for their rights,'' Ms. Cassidy said. They also help patients obtain treatment afterward and may eventually help with funeral arrangements and adoptions if there are children.
Ms. Cassidy works with a 26-year-old single mother of four who has AIDS. ''She is very isolated and desperate,'' Ms. Cassidy said. ''She didn't want anyone to know she has AIDS. What we've done is to be a real good friend.'' 'Still Such a Stigma'
Since the woman has no relatives who are willing to help out, Ms. Cassidy has been contacting adoption agencies to help place the children after their mother dies.
''What bothers me most is how AIDS is still such a stigma - to see somebody who is a kind, warm person, be treated badly by their families,'' Ms. Cassidy said. ''All of a sudden they get this disease and they're treated like lepers and it's very difficult to watch that.'' She spoke of a young woman with AIDS whose parents moved her to a back bedroom in their home and served her with paper plates and plastic spoons and forks. ''I'm always amazed at how many people are still terrified.''
Mr. Larson said, ''It seems so ironic and frustrating that people know how AIDS is transmitted and yet overreact in fear.''
Another volunteer, Gary Geter, said the most difficult thing about working with people with AIDS was ''hearing the malicious remarks, the scorn, the ridicule, the heartbreaking stories.'' 'I Am Euphoric'
Mr. Geter, a former intravenous drug abuser, said he could have been on the receiving end of the Buddy Program had he not straightened out his life 15 years ago. ''During my time, the worst I could have gotten was hepatitis,'' said Mr. Geter, who later attended college. ''I am euphoric that I had the good fortune to turn my life around.''
Besides being a Buddy volunteer, Mr. Geter, 38 years old, does outreach work in AIDS prevention for the Bridgeport Women's Project. One of his clients was a man whom he had known on the streets more than 15 years ago. They attended church services and watched football on television until the man died.
Volunteers can discuss their feelings and their work in a monthly support group that Ms. Cassidy runs. The sessions also give Ms. Cassidy a chance to sense whether they are becoming depressed or overburdened and need a respite.
There are waiting lists for both clients and volunteers, she said. There is insufficient money to run enough training sessions for additional volunteers. Each workshop costs $400 to $600, which covers guest speakers and refreshments. She said the next session would be in late August or mid-September.
''This has changed my life tremendously,'' Ms. Cassidy said of her work. ''It has helped me with my family and my children. They know the meaning of volunteering and they know about AIDS.
''I go to bed at night and I say, 'I feel real good about what I do with my day. I've accomplished a lot.' ''