6, 22, 31, 32 and 33 The study was conducted at the dedicated animal operation center of the Chinese PLA General Hospital, Beijing, China, with approval of the Animal Care and Use Committee. Thirty-four adult mongrel dogs of both sexes with an average body weight of 15 kg (range, 12-18 kg) were used. The canine model was chosen for its anatomic, physiologic, and immunologic similarity to humans.34 The animals were fasted from solid food LDE225 manufacturer for 48 hours before procedures but were allowed full access to water. All procedures were performed in a supine position with the animals under general anesthesia (pentobarbital

1 mg/kg, IM) and oxygen supplied after endotracheal intubation. A sterile forward-viewing, double working channel endoscope (2T200; Olympus Optical Ltd, Tokyo, Japan) inside an overtube was

inserted into the stomach followed by lavage of the stomach with 1000 mL 10% povidone-iodine solution through the working channel of the endoscope. The transgastric access site was located in the anterior gastric wall at the junction between the gastric body and antrum. A needle-knife sphincterotome (Boston Scientific Microvasive, Natick, MA) was used to create a 2-mm full-thickness incision, through which a guidewire was introduced and advanced into the peritoneal cavity. After dilatation of the incision site for 60 seconds with a 20-mm dilation balloon (CRE balloon, Boston Scientific Microvasive), both balloon and endoscope were advanced into the peritoneal cavity through the enlarged transgastric access. The animals were then subjected to an exploratory peritoneoscopy of 20 minutes and a gastrotomy http://www.selleckchem.com/products/nutlin-3a.html closure, after being randomly assigned into 1 of the 4 procedure groups

(see below) in either the survival or nonsurvival study. The survival and nonsurvival Bcl-w studies were carried out simultaneously. Endoscopic clips (HX-5LR-1; Olympus) were first applied to both ends of the incision to narrow the span of the gastric opening and then sequentially toward the center of the incision (Fig. 1A). The number of clips and time consumed for each closure were documented. The details of this procedure were described in the previous study.30 In brief, a free greater omentum flap near the serosal gastrotomy site was gently pulled into the gastric cavity by a pair of biopsy forceps. The omental flap was placed approximately 2 to 3 cm into the gastric cavity and then attached to the gastric mucosa with endoclips. All clips were positioned around the gastrotomy site to ensure effective sealing of the gastric defect approximately 1 to 2 cm away from the defects (Fig. 1B). No clips were deployed directly to close the gastrotomy site. After completion of the peritoneoscopy, the endoscope was removed and exchanged with a sterile single-channel upper endoscope (GIF 160; Olympus) mounted with a transparent applicator cap containing a modified 12-mm OTSC clip.

, 2005); GAD65 forward primer 5′-GAA CAG CGA GAG CCT GGT-3′, reverse primer 5′-CTC TTG AAG AAG CTC ATT

GG-3′ (362 bp expected band size); TPH2 forward primer 5′–TAA ATA CTG GGC CAG GAG AGG-3′, reverse primer selleck 5′-GAA GTG TCT TTG CCG CTT CTC-3′ (132 bp expected band size); and β-actin forward primer 5′-ATC CTG AAA GAC CTC TAT GC-3′, reverse primer 5′-AAC GCA GCT CAG TAA CAG TC-3′ (287 bp expected band size). The PCR conditions were conducted at 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 60–65 °C for 1 min, 72 °C for 1 min followed by 10 min at 72 °C. The PCR products were then separated in 1.5% agarose gel, stained with ethidium bromide, and then visualised under UV irradiation. Real-time RT-PCR amplification on a separate group of sham- (n=4) and NI-lesioned (n=4) was carried out using the ViiA 7 Real-time RT-PCR system (Applied Biosystems, USA) with SYBR green PCR master mix (Applied Biosystems, USA) with the following gene-specific

primers: CRF1 forward primer 5′-ACG ACA AAC AAT GGC TAC CG-3′, reverse primer 5′-GCA GTG ACC CAGGTA GTT GA-3′; relaxin-3 forward primer 5′–CCC TAT GGG GTG AAG CTC TG-3′, reverse primer 5′-CCA GGT GGT CTG TAT TGG CT-3′; GAD65 forward primer 5′-GGG GTG GAG GGT TAC TGA TG-3′, reverse primer 5′-ACC CAT CAT CTT GTG GGG AT-3′; TPH2 forward primer 5′-GCC TTT GCA AGC AAG AAG GT-3′, reverse primer 5′-CGC CTT GTC AGA AAG AGC AT-3′; and β-actin forward primer 5′-ATC CTG AAA GAC CTC TAT GC-3′, reverse primer 5′-AAC GCA GCT CAG TAA CAG TC-3′. β-actin was used as an internal control. The PCR conditions were an initial incubation

selleckchem of 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 59 °C for 1 min. Fresh NI/MS tissue was dissected and lysed with a radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0). Proteins were first quantified with a Pierce bicinchoninic acid assay (BCA) kit (Thermo Scientific, USA), separated on a 12% sodium dodecyl sulphate (SDS)-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 1% skim milk and incubated with a goat anti-CRF RI/II (1:1000, Santa Cruz, USA), rabbit anti-GAD65 (AB5082, 1:1000, Millipore, USA), rabbit anti-TPH2 aminophylline (1:1000, Chemicon, USA) and rabbit anti-actin (A2066, 1:10 000, Sigma-Aldrich, USA) or 5% Bovine Serum Albumin (BSA) solution and incubated with mouse anti-RLX3 (1:2500) overnight at 4 °C. The blot was then washed and incubated with a HRP-conjugated anti-goat (1:5000), anti-rabbit IgG (1:5000) or goat anti-mouse Alexa Fluor 488 (1:5000) for 1 h at room temperature with agitation. The proteins were then detected with a chemiluminescence kit.

If necessary, the filter can be applied several times; it operates by removing the magnetization of spins that reside

at the immobile site and therefore the diffusional decay detected at the end is, if the filter applied repeatedly, contributed only by those spins that resided on the “free” and mobile site during the whole diffusion time. In other words, the detected decay is supposed to be single-component with setting D = Df in Eq. (1). The pulse sequence with a single T2-filter was proposed previously [39] but without a detailed analysis, evaluation, and without having identified its possible use for eliminating exchange effects. The signal attenuation in the pulse sequence given in Fig. 2 can be found by analyzing the same set of coupled of differential PD0332991 purchase equations as above, Eq. (2a) and (2b).

The effect of the T2-filter is to re-establish after having applied the filter the same initial condition as in Eq. (6). As the other initial condition, at the end of the first τex delay and after having applied the first T2-filter, the free-pool magnetization is expressed similarly to that in Eq. (7a). equation(8a) Mz(q,τex)∝P′e-(2πq)2D1τex+(1-P′)e-(2πq)2D2τexMz(q,τex)∝P′e-(2πq)2D1τex+(1-P′)e-(2πq)2D2τex Hence, the BGB324 effect of any subsequent delay τex and T2-filter is to simply multiply the free-pool magnetization by the same factor; for n filters and thereby (n + 1) τex delays the obtained signal becomes equation(8b) S(q,n,τex)∝Mz(q,n,τex)∝(P′e-(2πq)2D1τex+(1-P′)e-(2πq)2D2τex)n+1S(q,n,τex)∝Mzq,n,τex∝P′e-(2πq)2D1τex+(1-P′)e-(2πq)2D2τexn+1

(We provide in Appendix A the formal solutions for those situations where delays τ1 and τrel are not of negligible length.) In that limit where the filter is applied with sufficiently high (τex ≪ 1/kb) frequency, the original exchange equation Eq. (2a) becomes modified by having suppressed any magnetization returning form the “bound” site equation(9) dMf(t)dt=-(2πq)2Df+kf+RfMf(t) As a result, the effect of exchange on the diffusional decay is removed and one retains the original Stejskal–Tanner expression with exchange almost solely exhibited as an intensity reduction equation(10) S=(S0e-kfΔ)e-γ2δ2g2(Δ-δ/3)DS=(S0e-kfΔ)e-γ2δ2g2(Δ-δ/3)Dby the factor exp(−kfΔ) that arises because longitudinal magnetization transferred to the “bound” site is eliminated. With τ1 ≫ T2b as is under consideration here, the system is selectively excited so that in the beginning of the τ2 period it is only the “free” site that exhibits nonzero longitudinal magnetization. This situation is similar to that explored in exchanging systems where spectral resolution permits the excitation of individual resonances by selective RF pulses [41]. As compared to conventional PGSTE experiments with nonselective RF pulses, the effect of exchange is reduced with selective excitation.

One of the most important reasons for clinicians needing a fast overview is when the record concerns a patient who is unknown [14]. We present here a computational system (a Report Generator) that automatically www.selleckchem.com/products/GDC-0941.html produces textual summaries of medical histories, and a study of its use by clinicians. We show that summaries, even when computer

generated, can be a useful tool for clinicians at the point of care, providing an accurate overview of the patient’s history in half the time. We developed a natural language generation system that produces a range of summarised reports of patient records from data-encoded views of patient histories derived from a repository of medical records of cancer patients, composed of narrative documents (e.g., letters, discharge reports, etc.) and structured data (e.g., test results, prescriptions, etc.) [20]. Although we are concentrating on cancer patients, we aim to produce good quality reports without the need to construct extensive domain models. Our typical user is a GP or clinician who uses electronic patient records at the point of care to familiarise themselves with a patient’s medical history and current situation. Information is extracted from medical narratives, using NLP techniques, as described in [21] and aggregated with structured data in order to build complex images of a patient’s medical

history which model the story of Osimertinib ic50 how the patient’s illnesses and treatments unfolded through time: what happened, when, what was done, when it was done, and why. The resulting complex semantic network, termed by us a Chronicle, allows the construction of targeted summarised reports which do more than present individual events in a medical history: they present, in coherent text, events that are Endonuclease semantically and temporally linked to each other. We provide here a brief general overview; more detailed technical descriptions of the Report Generator are available in [22] and [23]. The input to the Report Generator is a Chronicle. The methodology involved in transforming an EPR into a Chronicle is complex and involves

Information Extraction from narratives, solving multi-document coreference, temporal abstraction and inferencing over both structured and information extraction data [21]. The main advantage in using a Chronicle as opposed to a less structured Electronic Patient Record lies in the richness of information provided. Having access to not only facts, but to also the relations between them, has important implications in the design of the content selection and text structuring stages. This facilitates better and easier text generation and allows for a higher degree of flexibility of the generated text. The output of the Report Generator is a range of textual summaries of the information contained in the Chronology. These range in length from short paragraphs to many pages.

According to the Brazilian and the U.S. standards, the serving portion or RACC for individually packaged desserts is ½ cup (ANVISA, 2003a and US CFR, 2010b), which gives 120 g of product if milk-derived desserts are considered (ANVISA, 2003a). In this way, the limits imposed by such regulatory standards for the “low energy” claim behave quite similar for this kind of FK228 product. Considering the comparative

information for energy content, the claim “energy-reduced”, “light” or “lite” may be applied for products commercialized in Brazilian only if there is a reduction of 40 kcal per 100 g in the modified version of the solid or semi-solid food with the substitution of one or more ingredients, when compared to the original product (Brasil, 1998). For such a claim in the E.U., the energy value shall be reduced by at least 30% (EC, 2007), which are planned to be the same requirement to be adopted in Brazil (ANVISA, 2011). In the present study, this term could not be applied for energy according to the current and planned Brazilian standards, and the E.U. legislation (Table 7), once modified mousses did not attend the requisites when compared to control MF (Table 6). According to the U.S. legislation, “Light” or “Lite”

might be applied in two Pexidartinib in vitro situations: (1) if more than 50% of energy of a food product comes from fat and fat is reduced at least 50% compared to an appropriate food reference; or (2) if less than 50% of energy of a food product comes from fat and energy is reduced at least ⅓ per RACC or fat is reduced by 50% or more per RACC compared to a food reference (US CFR, 2010d). Considering the item number 2 (foods with less than 50% of their energy derived from fat) and the total fat content (reduction of fat higher than 50% compared to the original product), mousses I, WPC, I–WPC, and MF–I–WPC

could receive the “light” claim according to the U.S. legislation (Table 5, Table 6 and Table 7). In this case, this U.S. standard works with a different concept of energy reduction that seems to be more flexible, allowing a “light” claim to the products that are not able to attend this requisite according other standards. Low-fat” claim may be used in Brazil and in the E.U. for the absolute content of fat when any solid or semi-solid food presents a maximum Mannose-binding protein-associated serine protease content of 3 g of fat/100 g (Brasil, 1998 and EC, 2007). According to the U.S. legislation and that under planning to be adopted in Brazil, the claim “low fat” considers an absolute fat content of 3 g or less per RACC per eating occasion (½ cup) for a milk dessert product (ANVISA, 2011 and US CFR, 2010f). In Brazil, currently, when the modified version of a solid or semi-solid food is compared to its standard version in terms of fat, the claim “reduced fat” is allowed when a minimum reduction of 25% of fat and a difference above 3 g of fat/100 g is found between them (Brasil, 1998). In the U.S.

β-d-Salicin 1 and salicylic acid 2 are interesting phytochemicals that exert cross-biological AZD6244 mouse functions in plants and humans. This cross-function may be linked to the homological nature of DNAs in both plants and humans

and can be extended to animals and insects. In this respect, both cell regulatory proteins and nucleic acids, for example, possess the same amino acids or nucleotides repeating units, respectively. The match-up between a phytochemical and the corresponding receptor depend on the molecular recognitions and the stereo-compatibility of the interacted molecules. Therefore, mapping and analysing gene and expressed protein sequences of certain biosynthetic/pharmacologyical related pathways of certain phytochemical bioinformatically may contribute to devising new strategies in drug production. As such, β-d-salicin 1 and salicylic acid 2 may represent good examples in this respect, as both molecules exert biological activities in plants and humans to antagonise cell molecular

dysfunction. Author declare that there is no any conflict of interests. “
“Microbial electrochemical cells (MXCs) that include microbial fuel cells, microbial electrolysis cells, and microbial desalination cells show a promise selleckchem as sustainable wastewater treatment due to resource recovery (e.g., electric power, H2, CH4, water, H2O2, etc.). However, substantial energy loss in MXCs would trade off the profits of resource recovery, especially for large scale systems, and hence existing studies did not show clear benefits of MXCs, as compared to other anaerobic biotechnologies (e.g., anaerobic membrane bioreactors) [23]. In wastewater treatment perspectives, MXCs still have significant merit of no aeration requirement. Anode-respiring bacteria (ARB) that oxidize organic wastewater and transfer electrons to the anode in MXCs are anaerobes, which mean that MXCs can treat wastewater without significant

oxygen supply. Aeration costs account for 30–50% of operating and maintenance costs in municipal wastewater treatment facilities [33]. For instance, Tacrolimus (FK506) MXCs application to sewage treatment would save ∼$1.5 billion annually in Canada. To improve current density is crucial for MXC application to domestic wastewater treatment, since it represents wastewater treatability. Volumetric current density (A/m3 of anode chamber) is equivalent to organic loading rate (kg COD/m3 d), one of the most important design and operating parameters in wastewater treatment facilities. Organic loading rate typically ranges from 0.9 to 1.2 kg COD/m3 d in activated sludge [24] and [31], while it depends on the concentration of chemical oxygen demand (COD) in given domestic wastewater.

In PD, most biomarker discovery studies have relied on the proteome analysis of CSF. Using 2-DE, CSF profiling allowed the detection of a few differential proteins (i.e., complement c3) between control and PD patients [222] and [223]. Much more changes were detected in the CSF composition of PD patients using shotgun proteomic quantitative strategies as reviewed in [224]. Abdi et al.

found 72 proteins – including ceruloplasmin or apolipoprotein H, uniquely associated to PD compared to AD, dementia with LBs and control check details patient samples differentially labeled with iTRAQ-4plex [218]. Based on these results, Zhang et al. performed a large-scale validation of their best potential candidates using a Luminex assay and found that a panel of eight proteins (i.e., tau, amyloid β-42, β-2 microglobulin, interleukin- 8, vitamin D binding protein, apolipoproteins A-II and E and BDNF) was highly effective at identifying PD [225]. The selleck products proteomic analysis of plasma and serum

samples was proved challenging considering their complexity and the presence of a few highly abundant proteins. However, recent studies successfully highlighted potential PD biomarkers in blood [226], [227] and [228], of which the most promising may be plasma apolipoprotein A1 (ApoA1) [227]. This result was confirmed by independent studies based on multiplex and ELISA immunoassays, which suggested that low ApoA1 levels correlated with early PD onset and greater dopaminergic deficit as measured by putaminal DA transporter binding [229]. Alternatively, peripheral blood lymphocytes were investigated, highlighting a panel of five proteins (cofilin, tropomyosin, gamma-fibrinogen, ATP synthase beta and basic actin variant), which may be useful for PD diagnosis [230]. In the future, other sources of potential biomarkers accessible in

vivo may be investigated by proteomics ( Table 1, Table 3). Moreover, as shown on Fig. 1, tissue biomarkers may be found in peripheral regions susceptible to Lewy pathology such as submandibulary gland, colon, or skin [50], [53], [185] and [231]. These regions could be accessed through biopsy stiripentol in living patient and could allow the detection of early disease biomarkers, as the peripheral nervous system may be involved before the central nervous system in PD. Saliva was recently analyzed given its connection to the submandibular gland, which produces most of the salivary volume [29]. Importantly, α-SYN and DJ-1 were successfully identified in saliva, providing further relevance for the study of this fluid in a biomarker context [184]. Finally, unbiased proteomics investigations of post-mortem tissues from selected PD-relevant brain regions of neuropathologically confirmed cases might provide useful candidate biomarker proteins, which could further be screened in biofluids using immunoassay or targeted proteomics such as SRM.

Mehrabi et al.6, numa revisão compreensiva da literatura mundial, englobando 434 doentes, descreveu as várias

abordagens terapêuticas. Destes, particularizando, 128 doentes foram submetidos a transplantação hepática, com sobrevida global ao primeiro e terceiro anos de 96, e 54,5%, respetivamente. Assim, a transplantação hepática permanece como uma opção terapêutica razoável, mesmo em doentes com doença extra-hepática. mTOR inhibitor A sobrevida destes doentes é comparável à dos doentes transplantados por cirrose hepática22. Drogas antineoplásicas, tais como a talidomida, têm mostrado benefícios como terapêuticas adjuvantes ou como terapêutica inicial na doença irressecável23, assim como o interferon α-2 b24. A aplicação de quimioembolização transarterial poderá ser útil em doentes com doença hepática avançada, em lista de espera para transplante6, embora o seu papel como terapêutica coadjuvante no transplante hepático ainda não esteja

claramente determinado. Também doentes com localizações secundárias, tratados com esta modalidade terapêutica, parecem alcançar maior sobrevida do que Talazoparib solubility dmso com as modalidades cirúrgicas, poupando-os às suas comorbilidades 14. Há casos reportados de quimioterapia intra-arterial com redução do tamanho do tumor e sobrevida até 80 meses15. Os regimes quimioterapêuticos, de administração sistémica, estão longe de ser consensuais. Aqui, também múltiplos casos clínicos são reportados, utilizando múltiplos agentes, isolados ou em associação, com resultados díspares14, 25 and 26. Estas descrições apresentam-se sob a forma de casos clínicos, constituindo apenas casos descritivos, com falta Amine dehydrogenase de evidência constatada e de difícil interpretação, pela ausência de regimes terapêuticos uniformes e pela inexistência de estudos prospetivos. A maioria dos regimes de radioterapia (RT) foi administrada em conjunção com QT, pelo que as conclusões acerca da sua eficácia são problemáticas. Até ao momento, não estão determinados elementos prognósticos que permitam diferenciar os

doentes com HEH menos agressivos, dos indivíduos com uma doença mais agressiva, de forma a instituir uma atitude expectante ou, por outro lado, mais incisiva. Descrevemos o caso de um HEH manifestado num homem na quinta década de vida, sob a forma de desconforto no hipocôndrio direito. Apresentava-se já com um tumor de grandes dimensões, com suspeita de invasão pulmonar e óssea, e a evolução cursou com deterioração do estado geral de forma galopante, tendo falecido em menos de 2 meses após o diagnóstico. Este caso realça a imprevisibilidade do HEH, bem como a sua dificuldade diagnóstica em termos de imagem, principalmente na ausência do lollipop sign. Os autores declaram não haver conflito de interesses. “
“For decades, different cases of chronic pancreatitis associated with an important immunological component have been recognized. In 1961, Sarles et al.

The CRP preparation contained two very faint bands on either side of the 94 kD marker, in addition to CRP (Fig. 1b). These other proteins comprised less than 1% of the total protein and their presence is not surprising because several chromatographic steps required to remove traces of other proteins

in preparing the most highly purified CRP (de Beer and Pepys, 1982, Hawkins et al., 1991 and Carlucci http://www.selleckchem.com/products/Roscovitine.html et al., 2010) were not possible in the present pharmaceutical GMP procedure. The trace higher molecular weight proteins were identified by proteomic analysis, using mass spectrometry of trypsin digested fragments of the bands excised from the SDS‐PAGE. The lower mass band was the μ heavy chain of IgM and the higher mass band was plasmin/plasminogen (data not shown). The very faint trace bands

of mass lower than the SAP and CRP protomers are characteristic cleaved fragments of the protomers which are derived from intact pentameric pentraxins when they are reduced and denatured; they are invariably Talazoparib ic50 present in pentraxins isolated from ex vivo human material. No SAP was detected in the CRP preparation and no CRP was detected in the SAP preparation, which were tested at 3.0 and 1.5 mg/mL respectively using assays which in both cases detected the other pentraxin at 1 μg/mL ( Nelson et al., 1991 and Shine et al., 1981). The authentic covalent structures of the protomers of each pentraxin were confirmed by ESIMS, with average molecular masses (SD) (n = 3) of 25,462.64 (0.39) for SAP and 23,027.46 (0.52) for CRP, corresponding exactly to the predicted masses for their respective amino acid sequences, plus glycan in the case of 3-oxoacyl-(acyl-carrier-protein) reductase SAP (Pepys et al., 1994) and with N‐terminal PCA in CRP ( Oliveira et al., 1979). Integrity of the authentic native non‐covalent pentameric assembly of each protein was confirmed by gel filtration chromatography, which also showed the absence of any aggregation or dissociation into free protomers ( Fig. 2). The same result was obtained with the SAP preparation in non‐denatured gradient PAGE ( Fig. 3). Unlike the 4-30% gradient gels in Tris glycine we have previously used (

de Beer et al., 1982) but which are no longer available, human CRP does not form a discrete band in the present system. Functional activity of the proteins was confirmed by their reproducible, 100%, strictly calcium dependent binding to phosphoethanolamine-Sepharose beads (not shown). Furthermore these human proteins had the expected plasma clearance half life of ~ 3-4 h after intravenous injection into normal wild type C57BL/6 mice (Baltz et al., 1985 and Hawkins et al., 1988a) (shown for SAP in Fig. 4; not shown for CRP). This is a very sensitive test for structural and functional integrity of plasma proteins as even extremely subtle alterations, which may be undetectable by in vitro biophysical and biochemical methods, cause accelerated clearance of plasma proteins from the circulation in vivo.

The study was approved by Doxorubicin mouse the University of Cape Town’s Faculty of Health Sciences Research Ethics Committee and informed written consent was obtained from all volunteers before the study was initiated. Cervical cytobrush samples were collected according to the protocol described by Nkwanyana et al. (2009). Briefly, cervical immune cells were collected from all women under speculum examination by inserting a Digene cervical sampler into the endocervical os, rotating 360° and immediately placing the cytobrush in 3 ml R10 [RPMI

1640 (GibcoTM) supplemented with 5 mM glutamine, fungazone, penicillin, streptomycin and 10% FCS (Delta Bioproducts)]. Cytobrush samples with visible blood contamination (11/215; 5%) or excessive mucous contamination (21/215; 10%) were excluded from further analysis. Phenotypic and functional assessments of cytobrush-derived T cells were conducted in the remaining 183 samples. Samples were transported between the clinic and laboratory

in temperature-controlled click here benchtop coolers. Upon arrival in the laboratory (≤ 4 h of collection), the cytobrushes were flushed ~ 30 times with the same 3 ml transport media using a sterile plastic disposable Pasteur pipette and 25 ul of the suspension was removed for ex vivo CD3+ T cell enumeration using a Guava automated cell counter. The samples were divided into four groups to evaluate alternative processing conditions. Group 1 cytobrushes (n = 113) were processed immediately and used for flow cytometry analysis of immune subsets by intracellular cytokine staining (function, n = 98, Group 1a) and surface staining (viability, n = 15; Group 1b; ex vivo cytobrushes). Group 2 cytobrushes (n = 27) were not processed immediately but incubated at 37 °C for 24 h prior to flushing cells off the brush

and analysed for GNAT2 phenotype and function. Similarly, processing of cytobrushes from Groups 3 (n = 5) and 4 (n = 25) was delayed for 24 h and during this time, cytobrushes were maintained at 4 °C (to mimic cold overnight transport) or room temperature (~ 20 °C; to mimic overnight transport without refrigeration). After removing cervical cells off the cytobrush by gentle flushing, cells were washed once in R10, counted, phenotyped, and functionally evaluated using a Guava cell counter or FACS Calibur flow cytometer (BD Biosciences, San Jose, CA), respectively. Cervical cytobrush cells were counted using an automated Guava cell counter according to the method described by Nkwanyana et al. (2009). CD3-PE (T cells; Guava technologies) was used to label T cells in each cytobrush samples which were then counted using a Guava Automated Cell counter. Briefly, 25 μl cytobrush cells were stained with pre-titrated CD3-PE monoclonal antibodies and incubated at 4 °C for 30 min. Cells were washed with 1 ml wash buffer (1% FCS PBS) and centrifuged at 1500 rpm (437 ×g) for 5 min.