Among the others, IL-1 has been shown to be

a key cytokine in initiating and amplifying the inflammatory responses against H. pylori [37-39]. Very recently, IL-1β present in the gastric mucosa has been shown to play an important role in H. pylori-induced epigenetic changes linking inflammation to carcinogenesis [40]. Finally, H. pylori virulence and IL-1B genes contribute to peptic ulcers and intestinal metaplasia [41]. Elevation of Tregs at the site of infection and H. pylori-specific Tregs in the circulation [20, 21] has been suggested as a mechanism of pathogen persistence, on the assumption that Tregs are differentiated cells with professional suppressive function. In this study we show for the first time that H. pylori interacts with human Tregs indirectly via DCs and modifies their function. Our data show that H. pylori-treated DCs stimulate Treg proliferation, diminish their suppressive selleck compound see more function and that DC-derived IL-1β drives this process. Biopsy data from in-vivo H. pylori-infected antrum corroborated these findings, showing that a significant portion of Tregs found in infected gastric biopsies are actively undergoing mitosis. The persistence of H. pylori in the gastric mucosa may allow continual restimulation of the Treg population. This restimulation may allow for expansion of the Treg population beyond the 3-day peak observed in vitro. In this model it is not the presence of Tregs that promote the

Pomalidomide purchase persistence of infection, but rather the persistence of infection that expands the Treg population in an attempt to limit the damage caused by a prolonged and excessive inflammatory response. Demonstrations that suppressive function of Tregs can be undermined by pathogens have been shown previously in the context of L. major and H. hepaticus infections, limiting inflammation while hindering pathogen clearance [18, 19]. Although pathogens can influence Treg function directly, such as through engagement of TLR-2, -4 and -8 [42-44], we found that H. pylori had no direct effect on Tregs and that the changes induced in Treg behaviour could be explained by cytokine production from DCs. We have found that IL-1β plays a central role in mediating the effects of H. pylori on Tregs. This is of particular interest, as virulent strains of H. pylori expressing cagPAI are associated with elevated levels of IL-1β [13, 45]. As a result, the influence of H. pylori DCs on Tregs may be enhanced by the local microenvironment. In addition, IL-1β has a significant inhibitory effect on gastric acid production [46], which encourages H. pylori colonization to spread and downstream pathological events (gastritis and gastric cancer). As IL-1β appears to have a central role in H. pylori biology and its mechanisms of immune evasion and chronic inflammation, it may be revealing to study the relationship between polymorphisms in IL-1β and interactions between H.

trachomatis-infected cells in vitro (Rasmussen et al., 1997). Still, the fact that increases in MICA are see more seen only on infected cells but not on uninfected bystanders in the same culture suggests that soluble mediators are not sufficient for these effects. Chlamydia trachomatis infection mediates MHC class I downregulation

through direct mechanisms involving the degradation of the transcription factor, RFX5, by chlamydia protease-like activity factor (Zhong et al., 2000). We have previously demonstrated that ‘soluble factors’ could also mediate the downregulation of MHC class I (Ibana et al., 2011a). The downregulation of MHC class I by cytokines, including IL-10 (Caspar-Bauguil et al., 2000) and CXCL12 (Wang et al., 2008) has been demonstrated in other Ibrutinib solubility dmso culture models, supporting our previous observation that MHC class I downregulation occurs indirectly in the bystander-noninfected cells present in C. trachomatis-infected A2EN cells (Ibana et al., 2011a). Cytokine-mediated induction of dendritic cell MICA transcription by IFNα has been reported (Jinushi et al., 2003), but the overall effects of cytokines on MICA expression appear to be quite pleiotropic with varying effects depending on cell

type and environment (reviewed in Champsaur & Lanier, 2010). In the present study, we observed that MICA is upregulated only in infected cells, demonstrating that the mechanisms underlying C. trachomatis-associated changes in MICA differ from those Decitabine altering expression of MHC class I and suggesting C. trachomatis infection does not promote the production of soluble MICA-inducing mediators in our culture system. MICA was first described as cell stress-induced protein in the gastrointestinal epithelium (Groh et al., 1996). Increased MICA expression has been observed during both viral (cytomegalovirus) and

bacterial (M. tuberculosis) infections (Groh et al., 2001; Das et al., 2001). Our observation that upregulation of MICA was limited to C. trachomatis-infected cells may indicate that this induction is via infection-derived stress or danger signals that are absent in noninfected bystander cells. Currently, the exact mechanism underlying the induction of MICA expression during viral and bacterial infection is not completely understood. Interestingly, a recent study suggested that human microRNAs can regulate MICA expression, allowing the maintenance of MICA protein expression at a particular threshold while facilitating acute upregulation of MICA during cellular stress (Stern-Ginossar et al., 2008). If C. trachomatis infection induces MICA expression by interfering with the host microRNA-mediated control pathways, this may explain why MICA induction does not occur on uninfected bystander cells. The latter effect would protect the host from unwarranted NK cell activation.

11 Interleukin-32 is selectively expressed in activated natural killer cells, T cells, epithelial cells, endothelial cells and blood monocytes.11,12 The IL-32 induced by IL-18 has a number of splice variants, namely, IL-32α, -β, -γ, -δ, -ε and -ζ. Their receptors have yet to be identified, although proteinase 3 has been recently identified as a specific IL-32-binding protein. Interleukin-32 has emerged as

an important player in innate and adaptive immune responses13 and IL-32 associated with TNF-α appears to exacerbate TNF-α-related inflammatory arthritis and colitis.14 Expression of IL-32 may be a cancer biomarker, and high levels of IL-32 expression have been detected in several cancer cell types.15,16 Interleukin-32 knock-down was also shown to suppress anti-apoptotic proteins such as bcl-2, and induced apoptosis.15,17 Raf inhibitor Recent studies have

demonstrated that viral infection stimulates IL-32 expression; IL-32 suppressed replication of HIV18 during HIV infection, thereby reducing the levels of T helper type selleck compound 1 and pro-inflammatory cytokines,19,20 and was induced by infection with the influenza A virus.21 However, the role of HPV in IL-32 expression remains unclear. We detected IL-32 expression in tissues and cells obtained from patients with cervical cancer. Furthermore, as HPV plays a critical role in cervical cancer, we attempted to assess the possible role of IL-32 as an inducer of cancer and inflammation in response to HPV infection. Cyclo-oxygenase-2 (COX-2) is over-expressed in HPV-induced diseases, including cervical cancer,22,23 and is stimulated by HPV-16 E6 and

E7 Carbohydrate oncoproteins via the epidermal growth factor receptor/Ras/mitogen-activated protein kinase pathway.24 As COX-2 and IL-32 are associated with inflammatory processes, we attempted to characterize the relationship between COX-2 and IL-32 in the context of HPV infection. In this study, we evaluated the role of HPV in cervical cancer associated-IL-32 regulation as well as the feedback mechanisms between COX-2 and IL-32 occurring in response to the E7 oncogene. Human cervical cancer cells (C33A, SiHa and CaSki) were obtained from the American Type Culture Collection (Rockville, MD). An HPV-negative cervical cancer cell line (C33A) was prepared to establish stable cell lines expressing the E7 oncogene, and two stable cell lines (C33A/pOPI3, vector control C33A and C33A/E7, E7 expressing C33A) were established as previously described.25,26 All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and were cultured at 37° in a humidified atmosphere of 5% CO2. N-(2-cyclohexylosyl-4-nitrophenyl)-methane sulphonamide (NS-398) was purchased from Alexis Biochemicals (San Diego, CA), dissolved in DMSO, and used at final concentrations of 50 μm and 100 μm.

The level of significance was set at P = −0·05. In addition, linear Pearson correlation coefficients (r) were calculated using a linear regression model to measure the strengths and directions of the linear relationships between the number of TREC and the different age groups. These statistical analyses were performed using StatView (version mTOR inhibitor 5·0; SAS Institute, Inc., Cary, NC, USA). Graph were drawn using Microsoft Office Excel© or GraphPad Prism (version 4·0 for Windows; GraphPad Software, San Diego, CA, USA; http://www.graphpad.com). We were aware that any indication

of relative changes in sjTREC values in the samples could be compromised through a loss of integrity of the DNA. In order to ensure equivalence we analysed in excess of 250 samples and selected those for further analysis on the basis of their DNA integrity as determined by the amplifiability of the albumin gene [20,21]. Any sample with a Ct value greater than 24·0 cycles, which approximates to fewer than 1 × 105 albumin

molecules, was excluded from further analysis. Of the samples analysed approximately 17% were deemed unacceptable after albumin amplification, therefore we were able to identify 215 samples for further analysis. Surprisingly, a higher than expected proportion of unacceptable samples fell within the 80–89 age group, which is reflected as I-BET-762 molecular weight an apparent gap between 85 and 89 years. Analysis of the sjTREC per 105 T cells in our population (Fig. 1) showed a slow decline in their numbers between the 6th and 9th decade of life, with the most pronounced decline seen in those individuals more than 90 years of age. Inter-decade comparison of the sjTREC levels revealed that individuals in their 10th decade had significantly unless lower levels (P < 0·05) than

those obtained from individuals in the 7th, 8th and 9th decades (P-values of 0·0002, 0·0004 and < 0·0001, respectively). Moreover, samples from these earlier decades showed a wide range of values (see Table 1). Because of concerns that these results were due to changes either in the number of leucocytes or the number of CD3+ T cells in the blood of our donors [22] we analysed both of these parameters. Comparative analysis revealed no significant change across the age range (see Table 1), either in the number of leucocytes (P > 0·05) or in the absolute number of T cells (P > 0·05) as depicted in Fig. 2. Previous work has shown differences in sjTREC levels due to gender [23] The sex ratio measured in the present sample was near to 1, with approximately 52% (113 of 215) being females. In Fig. 3 the overall decline seen in both males and females highlights that females had higher levels of detectable sjTREC per 105 T cells compared to males at all age groups.

Given that the content of IgG in every reaction was 350-fold higher than that of H-gal-GP and that the antibody titres for the sera sources of pIgG were equivalent to those of npIgG [as shown by Smith et al. (9)], the experiment measures the true effect of H-gal-GP binding IgG from each source on haemoglobin digestion. Interestingly, whilst antibody inhibition of H-gal-GP catalysed haemoglobin digestion was detected at pH 5·0, no effect was seen if the complex and the antibodies were pre-incubated at pH 4·0 or 7·4 (even

though the antibodies bound to the H-gal-GP at both these pHs). Others working with Ancylostoma caninum also reported successful antibody inhibition PD0325901 order of protease activity. This inhibition was measured at pH 5·5 even though maximum rates of reaction were obtained under more acidic conditions at pH 3·5 (17,19–23). To our knowledge, the pH of the intestinal contents of Haemonchus has not been published, presumably because of the technical difficulties of obtaining a truly physiological sample. However, the reported pH of Schistosoma mansoni is between 6·0 and 6·4 (24,25). This would not be an optimal pH for protease digestion of blood proteins which operates most effectively under more acidic conditions. It has been suggested that these reactions may take place in luminal or cellular microenvironments which are more acidic or that the gradual decline in pH of the gut may be a mechanism by which

worms regulate the activity of each of these enzymes and hence the Selleck STA-9090 systematic degradation of blood proteins (26,27,28).

If the current Interleukin-3 receptor results accurately reflect what happens in vivo, it follows that optimum reaction conditions must exist within the Haemonchus gut to permit the specific inhibition of H-gal-GP by the antibody. The results generated by the present experiments support the hypothesis put forward in the introduction and suggest the following as the mechanism of protection in sheep immunized with H-gal-GP. Immunization with this antigen generates high titre circulating antibodies. When Haemonchus infect a sheep immunized with H-gal-GP, they ingest these antibodies with their blood meal. The antibodies inhibit the ability of H-gal-GP to digest haemoglobin and other blood proteins, leading to malnutrition and or starving of the parasites. The worms lay fewer eggs (9) and, being too weak to maintain their presence on the abomasal mucosa, get expelled through the pylorus by normal peristaltic activity. We thank David Knox and George Newlands for their academic input and Stephen Smith for technical assistance. “
“Colorado State University College of Veterinary Medicine & Biomedical Sciences, Fort Collins, CO, USA Cell & Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA, USA Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria Borrelia burgdorferi, the causative agent of Lyme disease, cycles in nature between a vertebrate host and a tick vector.

Proteomic studies of patient urine have identified exosomal fetuin-A as an early biomarker of acute Opaganib in vivo kidney injury,75 cleaved forms of β2-microglobulin as markers of acute renal allograft rejection,76 and a ubiquitin fusion protein (UbA52) as a potential specific marker of diabetic nephropathy.77 Interestingly, one of these studies also found that a fragment of degraded ubiquitin was specifically absent in urine from patients with diabetic nephropathy.77 Other researchers have focussed on urine

proteomic patterns as a means to predict the progression of kidney diseases with high sensitivity and high specificity. A urinary polypeptide pattern has been shown to distinguish IgA nephropathy from normal controls (90% specificity) and from patients with membranous nephropathy, minimal change disease, FSGS or diabetic nephropathy (100% specificity).78 Another urine proteomic study found that two proteins in a mass spectrometer signature can distinguish active and inactive lupus nephritis with 92% specificity.79 In addition, a clinical analysis has identified a CHIR-99021 mouse 12 peak proteomic mass spectrometer signature

that can predict cases of diabetic nephropathy in 74% of type 2 diabetic patients before the onset of microalbuminuria.80 Similarly, a more complex panel of 65 biomarkers Quisqualic acid has been shown to predict the development of diabetic nephropathy in patients with microalbuminuria (97% sensitivity) and differentiate from other chronic renal diseases (91% specificity).81 In this latter study, many of the urine biomarkers identified were fragments of collagen type I that were reduced in diabetic patients. One general concern with urine proteomic studies is that they can identify proteins as potential biomarkers when they have no known relationship to kidney injury, and this lack of connection to disease pathophysiology is a significant limitation.82 Recent advancements

in molecular analysis have resulted in the identification of a wide range of potential serum and urine biomarkers for assessing renal function and injury and predicting the development of kidney disease. Many of these biomarkers can be grouped according to their association with a particular type of injury (e.g. podocyte or tubular injury) or a mechanism of damage (e.g. oxidative stress, inflammation, fibrosis). Understanding the relationships between these different biomarker categories may help us to better understand disease processes. In addition, future assay developments may result in the creation of multiplex assays that target panels of biomarkers according to these specific categories.

In contrast, the 96-well plate format of the VeraCode-ASPE method enables HPV genotyping for large amounts of clinical samples. Furthermore, there are

a total of 144 different sets of VeraCode beads, and thus it is possible PF-562271 in vitro to include more HPV types in the VeraCode-ASPE genotyping format. In conclusion, the VeraCode-ASPE genotyping is a powerful new tool for the high-throughput HPV genotyping that will be required for large-scale surveillance of HPV-type distribution at the population level in the near future. This work received financial support from the Ministry of Health, Labor and Welfare in Japan, and the WHO HPV laboratory network. We thank Dr Roland Sahli at Centre Hospitalier Universitaire Vaudois in Lausanne for technical support for the introduction of the PGMY-RBH assay. The authors did not receive any financial support from the

companies whose products were used in this work. The authors have no conflict of interest to declare. “
“Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in C. difficile disease, see more but several surface-associated proteins of C. difficile are also recognized by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA-activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock Acesulfame Potassium proteins induced at 42 and 60 °C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins, and high levels of TNF-α, IL-1β and IL-8 were detected in response to challenge with culture supernatants. The

immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no interstrain difference observed in response to the culture supernatants of the tested C. difficile strains, but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B. Clostridium difficile is the causative agent of C. difficile disease (CDI; Bartlett et al., 1978; George et al., 1978). Previously associated primarily with the use of antibiotics and increasing age, today CDI is not uncommon in young, previously healthy adults with no history of antibiotic usage (McFarland et al., 2007). Although C.

8 mmol/L) and rapidly evolving acute

kidney injury due to acute tubular necrosis (ATN; initial creatinine 120umol/L, peak at 1210umol/L on day 4 post-diving accident). The diagnosis of ischaemia-induced ATN was supported by a high urinary fractional sodium excretion of 5.5%, elevated LDH (486U/L [125–250]) and a MAG3 scan in keeping with ATN. The absence of myoglobinuria and only moderately elevated creatine kinase (maximum 893U/L [30–170]) made rhabodmyoloysis-induced Sirolimus supplier ATN unlikely. He received supportive care with intravenous hydration, sodium bicarbonate and 100% oxygen followed by 7 sessions of hyperbaric therapy and recovered fully without needing dialysis. Conclusions: Arterial air embolism occurs when expanding gas ruptures alveolar capillaries (pulmonary barotrauma) and enters the arterial circulation as a result of rapid decompression. Clinical manifestations depend on the site of embolization and usually include neurological and respiratory symptoms but can also

involve the muscles, skin, mesenteric circulation and as shown in this case the kidneys. The diagnosis is made on clinical grounds since gas bubbles are rarely detectable on imaging. Best first aid for decompression illness is 100% oxygen therapy and supportive care but early transfer to a hyperbaric treatment unit is important as symptoms may evolve over time as in our patient. 277 HYPERKALEMIA INDUCES FAILURE OF PACEMAKER FUNCTION IN HEMODIALYSIS PATIENT N AUNG, S MAY Tamworth Base Hospital, New South Wales, Australia Background: Hyperkalemia may cause cardiac pacemaker selleck products malfunctioning due to a reduction of the electronegativity of the resting myocardial potential. Both sensing and capture mechanisms could be temporarily affected, with possible life-threatening effects. Case Report: Mr. DT, 50 years old male with background history of End Stage Renal Failure due to diabetic nephropathy on maintenance hemodialysis, Aortic Valve Replacement, Pacemaker for third degree AV block presented to ED in a small rural hospital with lethargy and unwell. BP 82/50 mmHg, HR 22/min. ECG showed significant bradycardia

20/min with failure of rhythm to capture the pacing. Arrangement was made for urgent transfer to Metropolitan unit with pacemaker malfunction. Subsequent Chlormezanone result: K 7.6 mmol, BSL 52.8 mmol. Repeat ECG show similar finding with no classic hyperkalemia changes. Patient was treated with usual medications for hyperkalemia and commenced on insulin infusion. At the same time, Haemodialysis was commenced. After 30 minutes on dialysis, patient’s vital sign improved to BP 100/70 mmHg, HR 65/min with ECG showing normal ventricular paced rhythm. Conclusions: Hyperkalemia is a cause of acute pacemaker malfunction without classical hyperkalemia ECG change due to a failure of pacemaker sensing and capturing. Acute treatment of hyperkalemia will restore pacemaker function.

Our findings demonstrate patency of the inferior epigastric vessels after ligation for TRAM delay during the

time frame usually used for delay to take effect. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“In this report, we present a case of treatment of fibrous dysplasia (FD) of the proximal femur with the pedicled iliac crest bone graft. An 18-year-old patient presented with hip pain and polyostotic dysplasia with involvement of the proximal femur and a history of pathological fracture. The patient was operated on using vascularized bone graft from the iliac crest and osteosynthesis with Dynamic Hip Screw (DHS®). With vascularized bone graft, we found an improvement on X-ray with no reabsorption, and with osteosynthesis, we controlled the pain and prevented pathological fracture and Selleck LY2835219 progression of the deformity. Several other studies where the pedicled iliac crest bone graft has been successfully used for the management of defects in the proximal femur (osteonecrosis of the femoral head and pseudarthrosis of the femoral head) can be found in the medical literature. However, the pedicled iliac crest bone graft in a patient with Selleck Poziotinib FD of the proximal femur is unique. © 2010 Wiley-Liss, Inc. Microsurgery, 2011. “
“Introduction: Restoring elbow flexion remains the

first step in the management of total palsy of the brachial plexus. Non avulsed upper roots may be grafted on the musculocutaneous nerve. When this nerve is entirely grafted, some motor fibres regenerate within the sensory fibres quota. Aiming potential utilization of these lost motor fibres, we attempted suturing the sensory branch of the musculocutaneous nerve onto the deep branch of the radial nerve. The objective of our study was to assess the anatomic feasibility of such direct suturing of the terminal sensory branch of the musculocutaneous Farnesyltransferase nerve onto the deep branch of the radial nerve. Methods: The study was carried out with 10 upper limbs from fresh cadavers. The sensory branch of the musculocutaneous muscle was dissected right to its division. The motor branch of the radial nerve was identified and dissected

as proximally as possible into the radial nerve. Then, the distance separating the two nerves was measured so as to assess whether direct neurorraphy of the two branches was feasible. Results: The excessive distance between the two branches averaged 6 mm (1–13 mm). Thus, direct neurorraphy of the sensory branch of the musculocutaneous nerve and the deep branch of the radial nerve was possible. Conclusions: When the whole musculocutaneous nerve is grafted, some of its motor fibres are lost amongst the sensory fibres (cutaneous lateral antebrachial nerve). By suturing this sensory branch onto the deep branch of the radial nerve, “lost” fibres may be retrieved, resulting in restoration of digital extension. © 2011 Wiley-Liss, Inc. Microsurgery, 2011.

[4-9] Hessell et al.[10] showed that an HIV-specific neutralizing learn more antibody mutated in the Fc position was no longer able to elicit Fc-mediated functions, such as ADCC, and that the efficacy in preventing simian/human immunodeficiency virus (SHIV) infection of macaques was significantly decreased, suggesting that the ADCC function is important for the protection afforded by neutralizing antibodies. There is a more limited understanding of the role of ADCC in the small subset of HIV-infected subjects who naturally control chronic infection, although the role

of cytotoxic T lymphocytes and neutralizing antibodies has been extensively studied.[11-24] We previously detected ADCC-mediated NK-cell activation in a small cohort of six subjects with slow HIV progression, but found no clear correlation with the magnitude of the ADCC response and control of LDE225 HIV. A recent study of 22 subjects indicated that elite controllers of HIV infection (subjects with consistent plasma HIV levels of < 50 copies/ml) have higher levels of ADCC antibodies than viraemic subjects, with an absence of correlation between cytotoxic T lymphocytes and neutralizing antibodies.[6] Whether these results are generalized across larger numbers of long-term slow-progressors

(LTSP) subjects is not clear. In addition, the HIV epitopes targeted by efficient ADCC are unknown but would logically be interesting vaccine targets. We analysed ADCC responses using an assay studying antibody-mediated interferon- γ (IFN-γ) and CD107a expression of NK cells. We

studied serum samples from 139 HIV-infected subjects not on anti-retroviral therapy; 65 subjects were LTSP who maintained a CD4 T-cell count of > 500/μl for at least 8 years after infection and the remaining 74 subjects were non-LTSP. We found that ADCC responses in LTSP subjects were broadly reactive against multiple HIV proteins and that LTSP subjects disproportionally targeted three specific ADCC epitopes within Vpu (viral protein U). The characteristics of the 139 subjects are shown in Table 1. All subjects were HIV-infected Bay 11-7085 and not on anti-retroviral therapy at the time of sampling. Subjects enrolled in both cohorts provided written informed consent and the relevant human research ethics committees approved all studies. Subjects were recruited both through the Long-term non-progressor network co-ordinated by the Kirby Institute, Sydney, Australia and through the Melbourne Sexual Health Centre, Australia. Sixty-five of the subjects met the pre-defined criteria as LTSPs, being HIV-positive for more than 8 years without anti-retroviral therapy and maintaining a peripheral CD4+ T-cell count above 500 cells/μl. There were no viral load entry criteria. The remaining 74 subjects did not meet the criteria for LTSP (i.e. had not maintained CD4 T-cell counts > 500 cells/μl for 8 years). For both cohorts, serum for ADCC testing was derived from the earliest time-point available.