In silico interrogation of the miRNAome of infected hematopoietic cells to predict processes important for human cytomegalovirus latent infection.

Human cytomegalovirus (HCMV) latency in CD34+ progenitor cells is the outcome of a complex and continued interaction of virus and host that is initiated during very early stages of infection and reflects pro- and anti-viral activity. We hypothesized that a key event during early infection could involve changes to host miRNAs, allowing for rapid modulation of the host proteome. Here, we identify 72 significantly upregulated miRNAs and three that were downregulated by 6hpi of infection of CD34+ cells which were then subject to multiple in silico analyses to identify potential genes and pathways important for viral infection. The analyses focused on the upregulated miRNAs and were used to predict potential gene hubs or common mRNA targets of multiple miRNAs. Constitutive deletion of one target, the transcriptional regulator JDP2, resulted in a defect in latent infection of myeloid cells; interestingly, transient knockdown in differentiated dendritic cells resulted in increased viral lytic IE gene expression, arguing for subtle differences in the role of JDP2 during latency establishment and reactivation of HCMV. Finally, in silico predictions identified clusters of genes with related functions (such as calcium signaling, ubiquitination, and chromatin modification), suggesting potential importance in latency and reactivation. Consistent with this hypothesis, we demonstrate that viral IE gene expression is sensitive to calcium channel inhibition in reactivating dendritic cells. In conclusion, we demonstrate HCMV alters the miRNAome rapidly upon infection and that in silico interrogation of these changes reveals new insight into mechanisms controlling viral gene expression during HCMV latency and, intriguingly, reactivation.

Provision of dietary advice for people with an ileostomy: a survey in the UK and Ireland.

AIM: The aim was to explore whether people with an ileostomy in the UK and Ireland receive the dietary advice they require. METHOD: An online survey with multiple-choice questions asked people with an ileostomy about the dietary advice they received and would have preferred to receive. Participants were recruited via websites of the Ileostomy and Internal Pouch Association and Crohn's and Colitis UK and via social media. People with a current ileostomy, age 16 years or over, and living in the UK or Ireland were eligible for inclusion. Responses were analysed using descriptive statistics. RESULTS: In all, 291 eligible responses were received and included in the analysis; 201 (69%) received advice on diet for their ileostomy from a healthcare professional or the internet. Of the 90 who did not receive dietary advice, 82 (91%) would have liked advice. Stoma nurses were the most common source of dietary advice (55%), but many other sources were frequently reported. Most (62%) felt that at least some dietary advice they received was conflicting. Over half (55%) felt anxious about managing their diet with a new ileostomy, 39% were confused, and 31% frustrated. Of 291 respondents, 29% received advice from a dietitian compared to 60% who would have preferred advice from a dietitian. CONCLUSION: Many people undergoing ileostomy surgery do not receive the dietary advice and support they require. Healthcare professionals working with people with an ileostomy should be mindful they are often anxious about their diet and require clear and consistent dietary advice and support.

Gonadotropin-inhibitory hormone in teleosts: New insights from a basal representative, the eel.

Since its discovery in birds, gonadotropin-inhibitory hormone (GnIH) has triggered investigation in the other groups of vertebrates. In the present study, we have identified a single gnih gene in the European eel (Anguilla anguilla), a representative species of a basal group of teleosts (Elopomorphs). We have also retrieved a single gnih gene in Osteoglossomorphs, as well as in more recently emerged teleosts, Clupeocephala. Phylogeny and synteny analyses allowed us to infer that one of the two gnih paralogs emerged from the teleost-specific whole genome duplication (TWGD or 3R), would have been lost shortly after the 3R, before the emergence of the basal groups of teleosts. This led to the presence of a single gnih in extant teleosts as in other vertebrates. Two gnih paralogs were still found in some teleost species, such as in salmonids, but resulting from the additional whole genome duplication that specifically occurred in this lineage (4R). Eel gnih was mostly expressed in the diencephalon part of the brain, as analyzed by quantitative real-time PCR. Cloning of eel gnih cDNA confirmed that the sequence of the GnIH precursor encoded three putative mature GnIH peptides (aaGnIH-1, aaGnIH-2 and aaGnIH-3), which were synthesized and tested for their direct effects on eel pituitary cells in vitro. Eel GnIH peptides inhibited the expression of gonadotropin subunits (lhß, fshß, and common a-subunit) as well as of GnRH receptor (gnrh-r2), with no effect on tshß and gh expression. The inhibitory effect of GnIH peptides on gonadotropic function in a basal teleost is in agreement with an ancestral inhibitory role of GnIH in the neuroendocrine control of reproduction in vertebrates.

Randomized clinical trial of postoperative chewing gum versus standard care after colorectal resection.

BACKGROUND: Chewing gum may stimulate gastrointestinal motility, with beneficial effects on postoperative ileus suggested in small studies. The primary aim of this trial was to determine whether chewing gum reduces length of hospital stay (LOS) after colorectal resection. Secondary aims included examining bowel habit symptoms, complications and healthcare costs. METHODS: This clinical trial allocated patients randomly to standard postoperative care with or without chewing gum (sugar-free gum for at least 10 min, four times per day on days 1-5) in five UK hospitals. The primary outcome was LOS. Cox regression was used to calculate hazard ratios for LOS. RESULTS: Data from 402 of 412 patients, of whom 199 (49·5 per cent) were allocated to chewing gum, were available for analysis. Some 40 per cent of patients in both groups had laparoscopic surgery, and all study sites used enhanced recovery programmes. Median (i.q.r.) LOS was 7 (5-11) days in both groups (P = 0·962); the hazard ratio for use of gum was 0·94 (95 per cent c.i. 0·77 to 1·15; P = 0·557). Participants allocated to gum had worse quality of life, measured using the EuroQoL 5D-3L, than controls at 6 and 12 weeks after operation (but not on day 4). They also had more complications graded III or above according to the Dindo-Demartines-Clavien classification (16 versus 6 in the group that received standard care) and deaths (11 versus 0), but none was classed as related to gum. No other differences were observed. CONCLUSION: Chewing gum did not alter the return of bowel function or LOS after colorectal resection. REGISTRATION NUMBER: ISRCTN55784442 (http://www.controlled-trials.com).

Patient experiences of perioperative nutrition within an Enhanced Recovery After Surgery programme for colorectal surgery: a qualitative study.

AIM: Nutrition is an important element of the Enhanced Recovery After Surgery (ERAS) programme. Patients have previously indicated that nutrition is a key component of ERAS that requires improvement. Our aim was to explore the perioperative nutrition experiences of colorectal surgical patients to identify barriers and facilitators to the integration of nutrition within ERAS. METHOD: Sixteen individuals undergoing colorectal surgery participated in a semi-structured interview between postoperative day three and hospital discharge. The topic guide was developed iteratively throughout the study; topics included preoperative counselling, carbohydrate loading, fasting and postoperative nutrition. A constant comparison technique was employed during coding, and an inductive thematic analysis was used. Validity was ensured by double coding a sample of transcripts. RESULTS: Findings are presented in the context of the following clinical themes: preoperative information, preoperative fasting, carbohydrate loading and nutritional drinks, postoperative diet and discharge. Individuals received too much general information which was repetitive, contradictory and not disease specific; this formed a key barrier affecting nutrition. Other barriers were negative experiences of nutritional drinks, stoma management, nausea and vomiting, and challenges from the hospital environment. Facilitators included interactions with staff, food accessibility and choice, and motivation for discharge. CONCLUSION: The key barrier to adherence of perioperative nutrition protocols was poor provision of information. Targeted information regarding postoperative diet, stoma management and coping with nausea and vomiting would be beneficial for colorectal surgical patients. Easily accessible food provided by ward staff was considered a facilitator.

Effects of donor/recipient human leukocyte antigen mismatch on human cytomegalovirus replication following liver transplantation.

BACKGROUND: Natural immunity against cytomegalovirus (CMV) can control virus replication after solid organ transplantation; however, it is not known which components of the adaptive immune system mediate this protection. We investigated whether this protection requires human leukocyte antigen (HLA) matching between donor and recipient by exploiting the fact that, unlike transplantation of other solid organs, liver transplantation does not require HLA matching, but some donor and recipient pairs may nevertheless be matched by chance. METHODS: To further investigate this immune control, we determined whether chance HLA matching between donor (D) and recipient (R) in liver transplants affected a range of viral replication parameters. RESULTS: In total, 274 liver transplant recipients were stratified according to matches at the HLA A, HLA B, and HLA DR loci. The incidence of CMV viremia, kinetics of replication, and peak viral load were similar between the HLA matched and mismatched patients in the D+/R+ and D-/R+ transplant groups. D+/R- transplants with 1 or 2 mismatches at the HLA DR locus had a higher incidence of CMV viremia >3000 genomes/mL blood compared to patients matched at this locus (78% vs. 17%; P = 0.01). Evidence was seen that matching at the HLA A locus had a small effect on peak viral loads in D+/R- patients, with median peak loads of 3540 and 14,706 genomes/mL in the 0 and combined (1 and 2) mismatch groups, respectively (P = 0.03). CONCLUSION: Overall, our data indicate that, in the setting of liver transplantation, prevention of CMV infection and control of CMV replication by adaptive immunity is minimally influenced by HLA matching of the donor and recipient. Our data raise questions about immune control of CMV in the liver and also about the cells in which the virus is amplified to give rise to CMV viremia.

Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands.

Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

Sex-specific mechanisms for eating disorder risk in men and women with autistic traits: the role of alexithymia.

BACKGROUND: A poorly understood relationship exists between eating disorders (ED) and autism spectrum conditions (ASC: henceforth 'autism'). ED are more prevalent in autistic people and people with high autistic traits, and autistic features are prognostic of longer illness. Aiming to understand what increases the risk of ED in relation to autism and autistic traits, previous research has implicated alexithymia as a causal mechanism in this relationship. These studies could not, however, disentangle whether alexithymia explains the relationship between ED pathology and autistic traits directly or through its impact on anxious/depressive symptoms, which in turn result in higher ED symptomatology. Moreover, despite evidence for sex differences in the aetiology of ED, little research has examined the impact of sex on these relationships. METHODS: Focusing on the association between autistic traits and ED psychopathology, we examined independent mediating effects of alexithymia and anxious/depressive symptoms, as well as sequential mediation effects where alexithymia affects ED psychopathology via its impact on anxious/depressive symptoms. Participants were 198 men and 265 women with formally diagnosed and suspected ED, who completed an online survey of standardised scales. RESULTS: In men, higher autistic traits were associated with ED psychopathology sequentially via greater alexithymia and through that, greater depressive/anxious symptoms. In women, alexithymia mediated the relationship between autistic traits and ED psychopathology both directly and sequentially through its impact on anxious/depressive symptoms. Interestingly, depressive/anxious symptoms also mediated that relationship independently from alexithymia. CONCLUSIONS: While cross-sectional, these findings suggest that the relationship between autistic traits and ED symptomatology is mediated by other variables. In support of its proposed role in the aetiology of ED, alexithymia was directly associated with ED symptoms in women. It also affected ED symptoms indirectly, in all participants, via its effect on depressive/anxious symptoms. Interventions focusing on alexithymia may facilitate recovery not only via their effect on ED, but via their effect on other forms of state psychopathology which contribute to the maintenance and development of ED. Sex differences, however, reflect that alternative therapeutic targets for men and women may be beneficial.

Autistic individuals seem to be at higher risk of developing eating disorders (ED)­even just having autistic traits seems to elevate risk of ED, although we do not understand why. One possibility is that autism and autistic traits are closely related to alexithymia, a difficulty identifying and describing your emotions, and it may be this that increases risk of ED. To test this, we explored relationships between autistic traits and ED symptoms in men and women with ED, who completed an online survey. In men, we found that autistic traits were associated with ED symptoms because they were associated with alexithymia, and alexithymia was associated with ED symptoms because it was associated with anxious/depressive symptoms. The same was true in women, but anxious/depressive symptoms were also associated with ED symptoms in their own right. While these findings need to be investigated in more thorough longitudinal approaches, they suggest that pathways to ED differ slightly between men and women, autistic and non-autistic, and that therapeutic interventions should also differ. In both sexes, the fact that alexithymia was associated with ED symptoms and those of other mental illnesses that seemed to contribute to ED, suggests that it should be targeted in interventions.

The cytomegalovirus gB/MF59 vaccine candidate induces antibodies against an antigenic domain controlling cell-to-cell spread.

Vaccination against human cytomegalovirus (CMV) infection remains high priority. A recombinant form of a protein essential for CMV entry, glycoprotein B (gB), demonstrated partial protection in a clinical trial (NCT00299260) when delivered with the MF59 adjuvant. Although the antibody titre against gB correlated with protection poor neutralising responses against the 5 known antigenic domains (AD) of gB were evident. Here, we show that vaccination of CMV seronegative patients induces an antibody response against a region of gB we term AD-6. Responses to the polypeptide AD-6 are detected in >70% of vaccine recipients yet in <5% of naturally infected people. An AD-6 antibody binds to gB and to infected cells but not the virion directly. Consistent with this, the AD-6 antibody is non-neutralising but, instead, prevents cell-cell spread of CMV in vitro. The discovery of AD-6 responses has the potential to explain part of the protection mediated by gB vaccines against CMV following transplantation.

Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy.

After allotransplantation, cytomegalovirus (CMV) may be transmitted from the donor organ, giving rise to primary infection in a CMV negative recipient or reinfection in one who is CMV positive. In addition, latent CMV may reactivate in a CMV positive recipient. In this study, serial blood samples from 689 kidney or liver transplant recipients were tested for CMV DNA by quantitative PCR. CMV was managed using preemptive antiviral therapy and no patient received antiviral prophylaxis. Dynamic and quantitative measures of viremia and treatment were assessed. Median peak viral load, duration of viremia and duration of treatment were highest during primary infection, followed by reinfection then reactivation. In patients who experienced a second episode of viremia, the viral replication rate was significantly slower than in the first episode. Our data provide a clear demonstration of the immune control of CMV in immunosuppressed patients and emphasize the effectiveness of the preemptive approach for prevention of CMV syndrome and end organ disease. Overall, our findings provide quantitative biomarkers which can be used in pharmacodynamic assessments of the ability of novel CMV vaccines or antiviral drugs to reduce or even interrupt such transmission.

Loss of caveolin-1 from bronchial epithelial cells and monocytes in human subjects with asthma.

BACKGROUND: Caveolin-1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation. METHODS: Therefore, we investigated whether caveolin-1 is deficient in asthmatic patients and in a murine model of asthma. RESULTS: Immunohistochemical analyses of endobronchial biopsies showed a remarkable loss of caveolin-1 in the lungs of asthmatic patients compared with controls. This loss was most evident in bronchial epithelial cells and associated with an increase in the expression of extracellular matrix proteins: collagen I, tenascin, and periostin. Cultured primary bronchial epithelial cells of asthmatics had lower caveolin-1 expression compared with control cells. In addition, caveolin-1 expression was significantly decreased in peripheral blood monocytes from asthma patients. The loss of caveolin-1 was also observed in a mouse model for asthma (mice sensitized and challenged with aspergillus fumigatus). CONCLUSIONS: To our knowledge, this is the first demonstration that the regulatory protein caveolin-1 is reduced in patients with asthma.

Percentage grade 4 tumour predicts outcome for prostate adenocarcinoma in needle biopsies from patients with advanced disease: 10-year data from the TROG 03.04 RADAR trial.

Previous reports have shown that quantification of high tumour grade is of prognostic significance for patients with prostate cancer. In particular, percent Gleason pattern 4 (GP4) has been shown to predict outcome in several studies, although conflicting results have also been reported. A major issue with these studies is that they rely on surrogate markers of outcome rather than patient survival. We have investigated the prognostic predictive value of quantifying GP4 in a series of prostatic biopsies containing Gleason score 3+4=7 and 4+3=7 tumours. It was found that the length of GP4 tumour determined from the measurement of all biopsy cores from a single patient, percent GP4 present and absolute GP4 were all significantly associated with distant progression of tumour, all-cause mortality and cancer-specific mortality over a 10-year follow-up period. Assessment of the relative prognostic significance showed that these parameters outperformed division of cases according to Gleason score (3+4=7 versus 4+3=7). International Society of Urological Pathology (ISUP) Grade Groups currently divide these tumours, according to Gleason grading guidelines, into grade 2 (3+4=7) and grade 3 (4+3=7). Our results indicate that this simple classification results in the loss of important prognostic information. In view of this we would recommend that ISUP Grade Groups 2 and 3 be amalgamated as grade 2 tumour with the percentage of GP4 carcinoma being appended to the final grade, e.g., 3+4=7 carcinoma with 40% pattern 4 tumour would be classified as ISUP Grade Group 2 (40%).

Associations between types of dietary fat and fish intake and risk of stroke in the Caerphilly Prospective Study (CaPS).

BACKGROUND: Few population-based longitudinal studies on diet and stroke have been conducted, and associations between dietary fat and fish intake and risk of stroke are unclear. OBJECTIVES: To prospectively examine relationships between intakes of total fat, saturated fat, unsaturated fat, white fish and oily fish and risk of stroke in a well-defined population of 2710 middle-aged men. STUDY DESIGN: Prospective cohort study. METHODS: Detailed information on health and lifestyle factors was collected via interview, and diet was assessed on three occasions using a food frequency questionnaire. Stroke ascertainment was by self-report and inspection of clinical records. Extracted data were assessed by two independent experts. RESULTS: During a median follow-up of 18 years, 225 strokes (209 ischaemic and 19 haemorrhagic) were eligible for inclusion in the analyses. For most recent diet (i.e. food frequency questionnaire data collected immediately prior to the stroke event), there was a slightly lower risk of stroke with higher intakes of unsaturated fat and oily fish. Multiple adjusted hazard ratios (HRs) for the lowest vs highest quintiles of unsaturated fat and oily fish intakes were 0.66 [95% confidence interval (CI) 0.41-1.05, P trend = 0.13] and 0.66 (95% CI 0.41-1.05, P trend = 0.09), respectively. Baseline and cumulative diets showed a slightly higher risk of stroke with higher intake of white fish; HRs for the lowest vs highest quintiles were 1.16 (95% CI 0.76-1.77, P trend = 0.22) and 1.28 (95% CI 0.77-2.13, P trend = 0.48), respectively. CONCLUSIONS: Overall, strong associations were not found between intakes of different types of fat and fish and risk of stroke in middle-aged men. The inverse associations between unsaturated fat and oily fish intakes and risk of stroke were weak, but the direction of association was broadly consistent with other studies; however, these relatively weak associations were not conventionally statistically significant.

Evaluation of urinary chloride dipsticks for the rapid estimation of hydration status in patients receiving artificial nutrition: Feasibility study.

BACKGROUND AND AIMS: The home parenteral nutrition (HPN) population face many challenges, especially with respect to fluid balance management. A low urinary sodium concentration of <20 mmol/L is commonly used as an indicator of dehydration that requires clinical assessment in these patients. The Quantab titrator dipstick measures chloride concentration of a solution and correlates with sodium concentration. We assessed whether it would be feasible to use the Quantab dipstick in the HPN population and explored relationships between Quantab dipstick estimated chloride concentration and quality of life (QOL). METHODS: Patients on HPN were asked to collect urine samples at 5 specific times points (day 0,7,14, 21 and 28) to send to the laboratory for formal electrolyte analysis. The participant and a member of laboratory staff tested these samples with the Quantab dipstick to estimate urinary chloride concentration. Participants were instructed to complete a QOL questionnaire at each of the 5 time-points in addition to a baseline demographic questionnaire and an end-of-study questionnaire. Six participants completed an interview at the end of the study period. The relationship between participant-derived and laboratory-derived data was assessed using rank correlation coefficients. QOL assessment was correlated with urine dipstick measurements. RESULTS: 10 patients on HPN completed the study. Data on chloride concentration as estimated by the dipstick (assessed by participants and by the laboratory) and sodium concentration from the laboratory were available for 47 urine samples. There was a positive relationship between participant dipstick estimated chloride concentration and laboratory sodium (Kendall's τ = 0.45; P < 0.001; Spearman's rs = 0.58 P < 0.001; 47 pairs). There was a strong correlation between chloride concentrations estimated by dipstick in the laboratory and by participants (Kendall 0.58 p < 0.001, Spearman's 0.69 p < 0.001; 47 pairs). In exploratory analyses, there was no relationship between QOL and dipstick estimated chloride concentration. Participants had no issues collecting urine samples but some difficulties were reported with determining the dipstick reading. CONCLUSIONS: Patients on HPN are able to collect urine specimens, complete QOL questionnaires, and are capable of using the Quantab dipstick to estimate urinary chloride concentration. The Quantab dipstick correlates with laboratory measured sodium and chloride concentrations. Further work is required to fully establish whether this point-of-care test could be used to guide fluid balance management in the HPN population.

Auditory processing deficits in children with reading and language impairments: can they (and should they) be treated?

Sixty-five children with specific reading disability (SRD), 25 children with specific language impairment (SLI), and 37 age-matched controls were tested for their frequency discrimination, rapid auditory processing, vowel discrimination, and consonant-vowel discrimination. Subgroups of children with SRD or SLI produced abnormal frequency discrimination (42%), rapid auditory processing (12%), vowel discrimination (23%), or consonant-vowel discrimination (18%) thresholds for their age. Twenty-eight of these children trained on a programme that targeted their specific auditory processing deficit for 6 weeks. Twenty-five of these 28 trainees produced normal thresholds for their targeted processing skill after training. These gains were not explained by gains in auditory attention, in the ability to do psychophysical tasks in general, or by test-retest effects. The 25 successful trainees also produced significantly higher scores on spoken language and spelling tests after training. However, an untrained control group showed test-retest effects on the same tests. These results suggest that auditory processing deficits can be treated successfully in children with SRD and SLI but that this does not help them acquire new reading, spelling, or spoken language skills.

Engorgement of Deep Medullary Veins in Neurosarcoidosis: A Common-Yet-Underrecognized Cerebrovascular Finding on SWI.

We describe the prevalence and potential significance of deep medullary vein engorgement on SWI in patients with neurosarcoidosis, a finding that has not been described previously. Engorgement was evaluated for possible associations with meningeal or perivascular disease, intracranial hemorrhage, and venous thrombosis, as well as with modified Rankin Scale scores at the time of MR imaging and at follow-up. Deep medullary vein engorgement was seen in 7 of 21 patients and was more common in men. Patients with venous engorgement had a significantly increased incidence of microhemorrhages, perivascular disease, and hydrocephalus. There was no association with the degree of leptomeningeal disease, venous dural sinus thrombosis, or modified Rankin Scale scores. In conclusion, deep medullary vein engorgement was common in our patients with neurosarcoidosis. Although its pathophysiology remains uncertain, it could be related to venous or perivenous abnormalities and may represent a useful secondary finding of cerebrovascular disease.

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin.

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

Compartmentalized dynamics of cytomegalovirus replication in treated congenital infection.

BACKGROUND: Cytomegalovirus (CMV) is the most prevalent congenital infection in developed countries. A significant number of infected infants develop long-term neurodevelopmental and hearing impairment irrespective of whether disease is detectable at birth. Studies of viral load and replication dynamics have informed the treatment of CMV in adult populations but no similar data exist in neonates. OBJECTIVES: To study CMV virus kinetics in different body fluids of babies treated for congenital infection. STUDY DESIGN: CMV virus load was sequentially analyzed in blood, urine and saliva in 17 babies treated for symptomatic congenital CMV infection. RESULTS: Virus was detectable in the urine and saliva of all babies at baseline but in only 15/17 in blood. At the end of 6 weeks of antiviral treatment CMV remained detectable in 9/14 blood samples, 9/12 urine samples and 4/7 salivary swabs. Median half-life (T1/2) of virus decline in blood was 2.4 days (IQR 1.9-3.3) and basic reproductive number (Ro) was 2.3. Although T1/2 values were similar in urine and saliva to those observed in blood, virus dynamics differed both during and after treatment. CONCLUSIONS: T1/2 and Ro in blood in this group of neonates were similar to values derived from studies of immunocompromised adults. The persistent viremia observed in treated neonates cannot therefore be adequately explained by the virus dynamics early in treatment. The different dynamics exhibited in blood and urine suggests that studying changes in distinct body compartments may assist in further understanding long-term manifestations of disease.

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen.

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.