, 2008). In our current studies, the HEp-2 cells were cocultured with the wild-type or the isogenic scl1-inactivated mutant GAS that were either treated or untreated with cFn or Lm. Following internalization, the numbers of surviving intracellular bacteria were determined. The Scl1-deficient GAS cells were internalized significantly less than click here the wild-type strain in ECM-free medium (Fig. 3). Following preincubation with cFn and Lm, the wild-type strain exhibited about a 4- and 6.5-fold

increase in internalization, respectively, compared with ECM-untreated cells. The scl1-inactivated strain preincubated with cFn and Lm also showed about a 2.2- and a 2.8-fold increase in internalization compared with the ECM-untreated mutant cells; however, the overall levels of mutant internalization were lower compared with the wild-type strain under each corresponding experimental condition, emphasizing the contribution of Scl1 to cell invasion by GAS. It should be noted that the in vivo relevance of GAS internalization by human cells mediated by ECM binding Pictilisib has been debated in recent years. In spite of this, recent investigations using nuclear magnetic resonance spectroscopy, circular dichroism analyses, and experiments with monoclonal antibodies identified structural changes caused by fibronectin upon binding to bacterial

proteins that result in an enhanced Fn recognition by integrins (Bingham et al., 2008; Margarit et al., 2009). It is tempting to speculate that Scl1 binding to cFn and Lm may exert similar biological effects. It was shown previously by our group

that Scl1 from M41-type GAS binds the human collagen integrin receptors, which mediates GAS internalization by host cells (Caswell et al., 2007, 2008a). Integrins bind the GLPGER sequence directly within the Scl1-CL region. Here, we show the V-region of the same Scl1.41 protein binds to cFn and Lm, which also increases GAS internalization by HEp-2 cells. We think it is unlikely that cFn and Lm binding to the globular V domain affects Scl1-CL region binding to α2β1 and α11β1; Nintedanib (BIBF 1120) however, we cannot fully exclude such a possibility. The HEp-2 cells express the α2, α3, α5, and β1 integrin subunits (Caswell et al., 2007), and are thus capable of producing the α2β1, α3β1, and α5β1 heterodimers with the ability to bind collagen, laminin, and fibronectin, respectively (Watt, 2002). The α11β1 integrin expression is restricted to fibroblasts (Popova et al., 2007) and, thus, may not be present on the surface of HEp-2 cells. Therefore, Scl1 may be contributing to internalization of M41-type GAS by HEp-2 cells by two mechanisms: direct binding to the α2β1 integrin and ECM-bridging mechanism via integrins α3β1 and α5β1.

The most commonly identified health problems were related to diabetes management, worsening of reflux or other chronic gastrointestinal complaints, difficulties with blood pressure control, exacerbation of mental health issues, and worsening of chronic pain complaints. Two patients required inpatient admission after return to the United States, one patient presented with a congestive heart failure exacerbation and the other with new-onset

atrial fibrillation in the setting of a hypertensive crisis. Both patients had been nonadherent Erastin concentration to antihypertensive medications during travel. By contrast, 34 patients (31%) reported a health problem that was new and not related to a chronic condition diagnosed prior to travel. Of these, 24 (22%) patients experienced an infection; most commonly, respiratory tract infections and skin and soft tissue infections. There were no reported hospitalizations in this group. A linear regression model using age of patient, duration of travel,

travel destination, number of medications before travel, documented nonadherence to medications, and whether chronic disease management was discussed as part of pre-travel counseling found that the number of medications Ion Channel Ligand Library taken before travel was associated with increased likelihood of a health problem related to a chronic condition. Patients were categorized as taking a small (0–3), moderate (4–6), large (7–10), or very large (>10) number of medications. For each increase in category, the odds of experiencing a health problem related to a chronic medical condition increased by 4.13-fold. A comparison of markers of chronic disease management before and after travel is described in Table 4. It did not reveal any statistically

significant changes, except for an average increase in DBP of 3.6 mmHg among patients with hypertension (p = 0.01). Subgroup analysis revealed that travel to Africa and reported nonadherence to medications were associated with worsening blood pressure PJ34 HCl control. Patients traveling to Africa experienced an increase in both SBP (131.8 ± 16 vs 138.1 ± 17.7, 95% CI [−12.87, 0.34]) and DBP (70.6 ± 10.4 vs 74.9 ± 8.7, 95% CI [−8.28, –0.39]) when values before and after travel were compared. Travel to Asia was not associated with worsening of blood pressure. Patients traveling to Africa also experienced a decrease in BMI (29.1 ± 2.8 vs 28.6 ± 3.3, 95% CI [0.04, 0.80]). Patients who were nonadherent to medications during travel, not surprisingly, also had an increase in both SBP (130.0 ± 16.3 vs 135.1 ± 17.8, 95% CI [−9.86, –0.56]) and DBP (69.2 ± 9.7 vs 73.2 ± 10.0, 95% CI [−6.45,–1.72]). On average, patients included in this study took the same amount of chronic medications before and after travel, 7 ± 4 medications. Sixty percent of patients reported nonadherence to one or more prescribed medications during travel.

The most commonly identified health problems were related to diabetes management, worsening of reflux or other chronic gastrointestinal complaints, difficulties with blood pressure control, exacerbation of mental health issues, and worsening of chronic pain complaints. Two patients required inpatient admission after return to the United States, one patient presented with a congestive heart failure exacerbation and the other with new-onset

atrial fibrillation in the setting of a hypertensive crisis. Both patients had been nonadherent AZD9291 to antihypertensive medications during travel. By contrast, 34 patients (31%) reported a health problem that was new and not related to a chronic condition diagnosed prior to travel. Of these, 24 (22%) patients experienced an infection; most commonly, respiratory tract infections and skin and soft tissue infections. There were no reported hospitalizations in this group. A linear regression model using age of patient, duration of travel,

travel destination, number of medications before travel, documented nonadherence to medications, and whether chronic disease management was discussed as part of pre-travel counseling found that the number of medications www.selleckchem.com/products/ldk378.html taken before travel was associated with increased likelihood of a health problem related to a chronic condition. Patients were categorized as taking a small (0–3), moderate (4–6), large (7–10), or very large (>10) number of medications. For each increase in category, the odds of experiencing a health problem related to a chronic medical condition increased by 4.13-fold. A comparison of markers of chronic disease management before and after travel is described in Table 4. It did not reveal any statistically

significant changes, except for an average increase in DBP of 3.6 mmHg among patients with hypertension (p = 0.01). Subgroup analysis revealed that travel to Africa and reported nonadherence to medications were associated with worsening blood pressure Niclosamide control. Patients traveling to Africa experienced an increase in both SBP (131.8 ± 16 vs 138.1 ± 17.7, 95% CI [−12.87, 0.34]) and DBP (70.6 ± 10.4 vs 74.9 ± 8.7, 95% CI [−8.28, –0.39]) when values before and after travel were compared. Travel to Asia was not associated with worsening of blood pressure. Patients traveling to Africa also experienced a decrease in BMI (29.1 ± 2.8 vs 28.6 ± 3.3, 95% CI [0.04, 0.80]). Patients who were nonadherent to medications during travel, not surprisingly, also had an increase in both SBP (130.0 ± 16.3 vs 135.1 ± 17.8, 95% CI [−9.86, –0.56]) and DBP (69.2 ± 9.7 vs 73.2 ± 10.0, 95% CI [−6.45,–1.72]). On average, patients included in this study took the same amount of chronic medications before and after travel, 7 ± 4 medications. Sixty percent of patients reported nonadherence to one or more prescribed medications during travel.

For each strain, one cosmid carrying hiC6 was analyzed by physical mapping and sequencing. For construction of physical maps,

cosmids were digested by single or double restriction enzymes, and the sizes of restricted fragments DNA Damage inhibitor were calculated based on their migration distances in agarose gel electrophoresis. hiC6 genes were localized to restriction fragments by PCR. For sequencing of the hiC6 region in the NJ-7 cosmid, a library of 2–4 kb Sau3AI DNA fragments (partial digestion) was constructed by insertion into the BamHI site of pUC19. hiC6-containing subclones were selected by PCR screening and sequenced. The sequence of the NJ-7 hiC6 region was assembled from overlapping subclone sequences. With the reference of the NJ-7 sequence, selleck screening library PCR fragments were generated for the hiC6 region of UTEX259 and sequenced. In addition, restriction fragments of this region in the UTEX259 cosmid were cloned and sequenced. The whole sequence of the UTEX259 hiC6 region was assembled from those of PCR and restriction fragments. In each case, the sequence was confirmed by

a series of PCRs using genomic DNA as the template. DNA sequences were deposited in the NCBI GenBank under accession numbers JF333588 (NJ-7 hiC6 genes) and JF333589 (UTEX259 hiC6 genes). Genomic DNA of C. vulgaris was extracted using the cetyltrimethylammonium bromide (CTAB) method (Murray & Thompson, 1980). A 10-μg aliquot of DNA was digested completely with one or two restriction enzymes. Separation of digested DNA with 0.7% agarose electrophoresis and capillary transfer of the separated DNA fragments onto Immobilon-Ny+ membrane (Millipore) were performed as standard methods (Sambrook et al., 1989). The digoxigenin (DIG)-labeled hiC6 probe for hybridization was prepared by PCR using hiC6-5 and hiC6-6 as primers and genomic DNA of NJ-7 as the template. Labeling,

hybridization and detection were performed with DIG High Prime DNA Labeling and Detection Starter either kit I (Roche) according to the manufacturer’s recommendations. Total RNA was extracted using Trizol reagent (Invitrogen) from C. vulgaris strains according to manufacturer’s instructions, separated by agarose/formaldehyde gel electrophoresis and blotted onto Immobilon-Ny+ membranes by capillary transfer. The hiC6 transcripts were probed by a PCR-generated 322-bp fragment overlapping the 3′-end of hiC6-3/4 cDNA (nt.380-701) of NJ-7. NJ-7 and UTEX259 were grown at 20 °C for 7 days and exposed to 4 °C for 24 h. Total RNA extracted from the algal cells with or without exposure to 4 °C was treated with RNase-free DNase I to remove residual DNA until no DNA could be detected by PCR, and then converted into cDNA using M-MLV reverse transcriptase (Promega). The transcription of each hiC6 gene was shown with RT-PCR with gene-specific primers listed in Supporting Information, Table S1.

For each strain, one cosmid carrying hiC6 was analyzed by physical mapping and sequencing. For construction of physical maps,

cosmids were digested by single or double restriction enzymes, and the sizes of restricted fragments Cisplatin price were calculated based on their migration distances in agarose gel electrophoresis. hiC6 genes were localized to restriction fragments by PCR. For sequencing of the hiC6 region in the NJ-7 cosmid, a library of 2–4 kb Sau3AI DNA fragments (partial digestion) was constructed by insertion into the BamHI site of pUC19. hiC6-containing subclones were selected by PCR screening and sequenced. The sequence of the NJ-7 hiC6 region was assembled from overlapping subclone sequences. With the reference of the NJ-7 sequence, MS-275 PCR fragments were generated for the hiC6 region of UTEX259 and sequenced. In addition, restriction fragments of this region in the UTEX259 cosmid were cloned and sequenced. The whole sequence of the UTEX259 hiC6 region was assembled from those of PCR and restriction fragments. In each case, the sequence was confirmed by

a series of PCRs using genomic DNA as the template. DNA sequences were deposited in the NCBI GenBank under accession numbers JF333588 (NJ-7 hiC6 genes) and JF333589 (UTEX259 hiC6 genes). Genomic DNA of C. vulgaris was extracted using the cetyltrimethylammonium bromide (CTAB) method (Murray & Thompson, 1980). A 10-μg aliquot of DNA was digested completely with one or two restriction enzymes. Separation of digested DNA with 0.7% agarose electrophoresis and capillary transfer of the separated DNA fragments onto Immobilon-Ny+ membrane (Millipore) were performed as standard methods (Sambrook et al., 1989). The digoxigenin (DIG)-labeled hiC6 probe for hybridization was prepared by PCR using hiC6-5 and hiC6-6 as primers and genomic DNA of NJ-7 as the template. Labeling,

hybridization and detection were performed with DIG High Prime DNA Labeling and Detection Starter Megestrol Acetate kit I (Roche) according to the manufacturer’s recommendations. Total RNA was extracted using Trizol reagent (Invitrogen) from C. vulgaris strains according to manufacturer’s instructions, separated by agarose/formaldehyde gel electrophoresis and blotted onto Immobilon-Ny+ membranes by capillary transfer. The hiC6 transcripts were probed by a PCR-generated 322-bp fragment overlapping the 3′-end of hiC6-3/4 cDNA (nt.380-701) of NJ-7. NJ-7 and UTEX259 were grown at 20 °C for 7 days and exposed to 4 °C for 24 h. Total RNA extracted from the algal cells with or without exposure to 4 °C was treated with RNase-free DNase I to remove residual DNA until no DNA could be detected by PCR, and then converted into cDNA using M-MLV reverse transcriptase (Promega). The transcription of each hiC6 gene was shown with RT-PCR with gene-specific primers listed in Supporting Information, Table S1.

During 2005–2007, a third of women delivered vaginally, half by elective CS and the remainder by emergency CS. In contrast, at the start of the HAART era, two-thirds of women delivered by elective CS. We document geographical variation in mode of delivery in the HAART era, with an increasing proportion of vaginal deliveries, mainly in the United Kingdom, Belgium and the Netherlands. In multivariable analysis of MTCT risk among MCPs with maternal HIV

RNA <400 copies/mL, elective CS was associated with an 80% decreased MTCT risk. However, among women with viral loads <50 copies/mL there were only two transmissions overall. Although clinical trials are the gold standard for clinical care, observational studies often provide initial evidence for trial inception and design. Use of elective CS Apoptosis Compound Library high throughput SCH772984 concentration as a PMTCT intervention is a case in point: the ECS first published results showing an association between reduced MTCT risk and elective CS in 1994 [5],

with subsequent confirmation from a large meta-analysis [9]. Our finding here that the peak elective CS rate occurred in 1999, when the mode of delivery trial was published [8], is probably largely explained by participating clinicians changing their practices before the trial results were released based on the observational evidence they helped to provide; furthermore, some women were concomitantly enrolled in both the trial and the ECS. The somewhat paradoxical finding of a declining elective CS rate in the years immediately following the trial publication may be partly explained by the concurrent implementation of antenatal HAART

instead of mono- or dual therapy for PMTCT, when the first studies suggesting the benefit of HAART for decreasing MTCT risk were published [24–27] and guidelines started to change. In the Netherlands, for instance, the national guideline in 2000 only mentioned an elective CS as a rescue therapy in case of HAART failure or refusal [28]. Other European studies have also documented declining elective CS rates in the HAART era. In an analysis from the French Quisqualic acid Perinatal Study involving over 5000 pregnant women receiving antenatal ART and delivering between 1997 and 2004, the elective CS rate declined from 56% in 2000 to 41% in 2004 [4]. In the United Kingdom and Ireland National Study of HIV in Pregnancy and Childhood (NSHPC), the elective CS rate peaked in 1999 at 66%, declining to around 50% in 2006. The emergency CS rate we report here was relatively stable but high and ranged from 15% to 17% in the HAART era; the French Perinatal Study also reported stable emergency CS rates between 1997 and 2004, but higher at around 29% [4].

Both succinate and NADH caused fluorescence quenching, which was eased after the addition of an uncoupler, proving that the observed quenching was indeed caused by the PMF (Fig. 1a). Quenching reached a maximum after ∼10 min (Fig. 1a), significantly slower than IMVs from Escherichia coli under identical conditions (data not shown). This slow quenching may be caused by a larger percentage of leaky IMVs. The lower PMF observed with NADH (11% quenching) compared with succinate (39%) might be due to the partial detachment of the membrane-associated type-II NADH-dehydrogenase

(NDH-II), the main NADH-oxidizing enzyme of the respiratory electron transport chain in M. bovis BCG (Boshoff RG7422 mouse et al., 2004; Weinstein et al., 2005). From these results, it can be concluded that the IMVs are functional. Similarly, IMVs from the fast-growing M. smegmatis accepted both NADH and succinate as electron donors (Fig. 1b). We then investigated whether the IMVs can establish a PMF with www.selleckchem.com/products/atezolizumab.html ATP as a substrate. No significant quenching was detected either for M. bovis BCG or for M. smegmatis, even after an extended (>30 min) incubation time (Fig. 1a and b). The very small intensity decrease directly after ATP addition is due to sample volume increase and is not reverted with the addition of an uncoupler. Neither variation of the ATP/Mg2+ ratio

(from 0.4 : 1 to 2 : 1), or variation of the pH value (pH 5.5–8.0) nor preparation of IMVs

from M. bovis BCG cultured in an oxygen depletion model (Wayne system) led to detectable quenching upon ATP addition. These results indicate that mycobacterial ATP synthase is not carrying out ATP-hydrolysis-driven proton transport. To exclude the possibility that the observed lack of ATP-hydrolysis-driven Megestrol Acetate proton transport is caused by an extremely low number of ATP synthase molecules in the mycobacterial membrane or by of the detachment of the extrinsic F1 part of ATP synthase, we compared the DCCD-sensitive activities in ATP synthesis and ATP hydrolysis. As shown in Table 1, the IMVs from both M. bovis BCG and M. smegmatis were active in ATP synthesis with specific activities of 0.27 and 0.96 nmol min−1 mg−1, respectively. In contrast, we could not detect any significant DCCD-sensitive ATP hydrolysis activity in IMVs from M. bovis BCG. For M. smegmatis IMVs, DCCD-sensitive ATP hydrolysis activity was detectable, but >4-fold lower as compared with ATP synthesis (Table 1). For an enzyme working with equal speed in both directions, the ATP hydrolysis activity is expected to be higher than the synthesis activity, for example ∼10-fold for ATP synthase from Bacillus PS3 (Bald et al., 1998, 1999). This effect is due to the presence of enzymes in leaky vesicles, unavoidably present in IMV preparations, which can split ATP, but are unable to synthesize it.

The EACS and BHIVA guidelines have a similar approach in relation to the threshold for screening for risk of fractures. Both recommend that patients should only be considered for screening with DXA scans when there is a significant risk. The BHIVA guidelines

define this as those with an intermediate or high FRAX score, and all men and women over 70 and 65 years of age, respectively. BHIVA guidelines recommend that a 3-yearly assessment of risk factors becomes Panobinostat nmr relevant at 50 years of age or above, and should be additionally performed for this age group before and after the use of ART. EACS guidelines suggest a 2-yearly follow-up in those > 40 years of age, again reserving DXA for those considered to have significant risk. Both guidelines focus on specific time-points relating to HIV therapy: baseline (the point at which the patient engages in care); pre-ART initiation; at ART initiation; on ART (6–12-monthly for most comorbidities, except for bone in which

the gradual nature of the change allows for screening on average every 2–3 years). Regular screening would identify those HIV-infected individuals most at risk of developing metabolic comorbidities and means that appropriate interventions can be initiated to reduce modifiable risk factors. Lifestyle interventions will be adequate for most individuals. Pharmacological management is indicated for the minority, but for these, the potential risk reduction can be large, with a commensurate mitigation of morbidity and mortality. Table 1 summarizes the assessments and treatment recommendations Apoptosis Compound Library in vitro for noninfectious comorbidities in the current EACS guidelines. + + + + + + +/ + + + + Annual 3–12 months + + + + + + Annual 3–12 months Annual + + + + + 6–12 months 2 years As indicated 1–3 years 1–3 years 1–3 years 6 months ALP, alkaline phosphatase; ALT, alanine amino transferase; aMDRD, abbreviated modification of diet in renal disease; ART, antiretroviral therapy; AST, aspartate amino transferase; CKD, chronic

kidney disease; CVD, cardiovascular disease; DXA, dual energy Sunitinib solubility dmso X-ray absorptiometry; ECG, electrocardiogram; eGFR, estimated glomerular filtration rate; FBC, full blood count; FRAX, fracture prediction tool; G6PD, glucose 6-phosphate dehydrogenase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; MSM, men who have sex with men; PI, protease inhibitor; TC, total cholesterol; TG, triglycerides; UP/A, urine protein/albumin ratio; UP/C, urine protein/creatinine ratio. HIV infection may contribute to an increase in cardiovascular risk through several potential mechanisms, including increased systemic inflammation, pro-atherogenic changes in serum lipids, increased systemic hypercoagulability and decreased vascular reactivity [29].

Fourteen cohort studies provided information on causes of death and were included in analyses presented in this paper. All studies that joined the collaboration have been approved by their local ethics committees or institutional selleck chemicals llc review boards, use standardized methods of data collection, and schedule follow-up visits at least once every 6 months. Patient selection and data extraction were performed at the

data centres of the participating cohort studies. Anonymized data from each cohort on a predefined set of demographic, laboratory and clinical variables were pooled and analysed centrally. Data managers checked for duplicated records, and ensured that patients included in more than one cohort had only one record in the combined data set. The primary endpoint in this study was HIV disease progression, defined as (1) a new AIDS-defining disease [based on the clinical part of the 1993 US Centers for

Disease Control and Prevention (CDC) revision of the AIDS case definition] or (2) death from any cause. We utilized an intent-to-continue-treatment approach, and therefore ignored changes to treatment regimen, including treatment interruptions Osimertinib chemical structure and terminations. We measured time from the initiation of cART to the date on which the endpoints occurred. Patients who remained alive were censored at their last visit plus 50% of the average time between visits for that cohort. For example, if a cohort had, on average, 6 months between follow-up visits, patients who did not die would be censored at last visit plus 3 months. This allocates follow-up time in an unbiased way to those who did not die, as the average time from last follow-up to death in those who died is approximately 50% of the interval between scheduled visits.

The secondary outcomes in this study were causes of death. All deaths with International Classification of Diseases (ICD) version 9 or ICD10 or free text coding were reviewed by a computer program and also by a clinician and an Protein Tyrosine Kinase inhibitor epidemiologist and then reviewed in committee when discordant. Cause of death was determined utilizing a standardized protocol developed by the Copenhagen HIV Programme for coding causes of death in HIV-positive individuals [25]. Two cohorts participating in ART-CC [Italian Cohort of Antiretroviral-Naïve Patients (ICONA) and the Veterans Aging Cohort Study (VACS)] did not provide causes of death and were omitted from analyses. The two cohorts from Germany did not provide cause of death prior to 2002 for patients in Frankfurt and prior to 2003 in Cologne and Bonn clinics. Patients enrolled in these cohorts prior to these years were excluded.

bulgaricus (ATCC 11842) (Christian www.selleckchem.com/products/abt-199.html Hansen A/S, Denmark) and L. rhamnosus GG (ATCC 53013) (a kind gift from Dr Seppo Salminen of University of Turku, Finland) were streaked onto deMan Rogosa Sharpe agar (Difco Laboratories) and incubated at 37 °C in 5% CO2. Single colonies were

used to produce seed cultures (9 h), which were used to initiate 50-mL cultures. Bacteria were harvested at the late log phase (OD550 nm for L. casei, L. rhamnosus and L. bulgaricus were 5.7, 8.8 and 8.6, respectively, and the CFU were approximately 2 × 109, 1 × 109 and 3 × 109 mL−1, respectively) by centrifugation at 1699 g for 10 min at room temperature and washed twice with sterile saline (0.85% NaCl). The CFU were determined by plating serial dilutions of the bacterial GSI-IX research buy samples on deMan Rogosa Sharpe agar plates that were incubated at 37 °C in 5% CO2. Lyophilized bacteria were prepared by freezing bacterial pellets (−80 °C for at least 9 h) that were washed with saline before overnight lyophilization in a freeze-dryer at −40 °C, vacuum pressure 400 mbar (Thermo Savant). Lyophilized bacteria were stored at −80 °C. The viability of the preparations was about 6%. All animal studies were conducted according to the Institutional Guidelines at the National University of Singapore. Spleens

isolated from female C57BL/6 or BALB/c mice (4–6 weeks old) were cut into small pieces and treated with 2 mg mL−1 collagenase (Sigma-Aldrich) in Roswell Park MG-132 cell line Memorial Institute (RPMI) 1640 medium for 25 min at 37 °C. The spleen pieces were further separated with a plunger of a 1-mL syringe and filtered through a 70-μm cell strainer (BD Falcon). The cell suspension was centrifuged at 453 g for 5 min and the pellet was resuspended in 1 mL of red blood cell lysis buffer (150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA) and incubated at room temperature for 5 min. The cells were centrifuged at 453 g for 5 min, washed with phosphate-buffered saline (PBS) twice and finally resuspended in RPMI 1640 medium

supplemented with 10% fetal bovine serum (Hyclone), 2 mM l-glutamine (Gibco, Japan) and 50 μg mL−1 Penicillin G-Streptomycin (Sigma-Aldrich). The spleen cells were plated at a density of 2 × 106 cells per well in a 24-well plate (Nunc, Denmark) and cultured with either L. rhamnosus, L. bulgaricus or L. casei (2 × 108 CFU per well) at 37 °C for 6, 24, 48 and 72 h in 5% CO2. The coculture experiments were performed in the presence of antibiotics to prevent overgrowth of the bacteria and a decline in pH as a result of lactic acid production. The pH of the supernatants was monitored. Lyophilized lactobacilli in general induced a smaller decline in pH (0.1–0.2) compared with live bacteria (0.4–0.6) probably due to the low viability of these preparations. The lowest pH monitored was between 6.6 and 6.7 in cells treated with live bacteria and this is not expected to affect cytokine stability.