Role of Microfiltration Membrane Morphology on Nanoparticle Purification to Enhance Downstream Purification of Viral Vectors.

In the rapidly advancing realms of gene therapy and biotechnology, the efficient purification of viral vectors is pivotal for ensuring the safety and efficacy of gene therapies. This study focuses on optimizing membrane selection for viral vector purification by evaluating key properties, including porosity, thickness, pore structure, and hydrophilicity. Notably, we employed adeno-associated virus (AAV)-sized nanoparticles (20 nm), 200 nm particles, and bovine serum albumin (BSA) to model viral vector harvesting. Experimental data from constant pressure normal flow filtration (NFF) at 1 and 2 bar using four commercial flat sheet membranes revealed distinct fouling behaviors. Symmetric membranes predominantly showed internal and external pore blockage, while asymmetric membranes formed a cake layer on the surface. Hydrophilicity exhibited a positive correlation with recovery, demonstrating an enhanced recovery with increased hydrophilicity. Membranes with higher porosity and interpore connectivity showcased superior throughput, reduced operating time, and increased recovery. Asymmetric polyether sulfone (PES) membranes emerged as the optimal choice, achieving ∼100% recovery of AAV-sized particles, an ∼44% reduction in model cell debris (200 nm particles), an ∼35% decrease in BSA, and the fastest operating time of all membranes tested. This systematic investigation into fouling behaviors and membrane properties not only informs optimal conditions for viral vector recovery but also lays the groundwork for advancing membrane-based strategies in bioprocessing.

Lateral tarsal strip plus skin-muscle flap excision in the treatment of lower eyelid involutional entropion.

This article examines the effectiveness of skin-muscle flap excision in conjunction with a lateral tarsal strip for the treatment of involutional entropion. Ninety-six eyelids in 83 consecutive patients with involutional entropion were treated using a standardized surgical procedure. All patients underwent lower eyelid tightening with a lateral tarsal strip, dissection of a skin-muscle flap inferiorly through a subciliary incision and excision of redundant skin as well as orbicularis muscle. Follow-up data was obtained by retrospective chart review and telephone interviews. 80 patients were included in this study. The only exclusion criteria was failure to attend the 1 week follow-up appointment, n = 3. There was only one recurrence which was mild and revised under local anesthesia. Two patients had overcorrection with mild ectropion but did not require additional surgery. In those that completed their initial post-operative visit, the average time follow-up time was 502 days. Excision of a skin-muscle flap is a useful addition to lateral tarsal strip surgery in the treatment of involutional entropion and is a quick procedure producing excellent functional and cosmetic results. To our knowledge, this is the first cohort of patients to be reported using this technique where all patients had a standardized surgical approach. Additional studies are needed to compare long-term outcomes of this technique against other surgical treatments.

Active Choice and Financial Incentives to Increase Rates of Screening Colonoscopy-A Randomized Controlled Trial.

Behavioral economic approaches could increase uptake for colorectal cancer screening. We performed a randomized controlled trial of 2245 employees to determine whether an email containing a phone number for scheduling (control), an email with the active choice to opt in or opt out (active choice), or the active choice email plus a $100 incentive (financial incentive) increased colonoscopy completion within 3 months. Higher proportions of participants in the financial incentive group underwent screening (3.7%) than in the control (1.6%) or active choice groups (1.5%) (P = .01 and P < .01). We found no difference in uptake of screening between the active choice and control groups (P = .88). The $100 conditional incentive modestly but significantly increased colonoscopy use. ClinicalTrials.gov no: NCT02660671.

Accuracy and patient-centered outcome variables in guided implant surgery: a RCT comparing immediate with delayed loading.

AIM: To assess the accuracy and patient-centered outcome of a novel guided surgery system for placing implants in an edentulous maxilla. MATERIAL AND METHODS: Fifteen consecutive patients with sufficient bone to place six implants in the maxilla were randomly assigned to the immediate loading (with delivery of the final prosthesis within 24 h) or the delayed loading treatment group. Accuracy was assessed by matching the planning CT with a postoperative CBCT. Patient-centered outcome measures were the Dutch version of the McGill Pain Questionnaire (MPQ-DLV), the health-related quality of life instrument (HRQOL), visual analog scales (VAS), the duration of the procedure, and the analgesic doses taken each day. RESULTS: A mean deviation was found at the entry point of 0.9 mm (range: 0.1-4.5, median 0.8) and of 1.2 mm (range: 0.2-4.9, median 1.1) at the apex, and an angular deviation of 2.7° (range: 0.0-6.6°, median 2.3) was observed. The mean vertical deviation was 0.5 mm (range: 0.0-3.2, median 0.4), and in a horizontal direction, this was 0.7 mm (range: 0.1-3.1, median 0.6). The mean deviation in mesio-distal direction was 0.5 mm (range: 0.0-2.3, median 0.4) and in bucco-lingual direction 0.5 mm ± 0.4 (range: 0.0-2.2, median 0.3). No statistical differences could be shown between treatment groups on pain response (MPQ-DLV), treatment perception (VAS), number or kind of pain killers, or for the HRQOLI instrument. CONCLUSION: The accuracy of a novel CT-based guide is comparable to the accuracy data of other systems. Within the limitations of this study, no difference could be found in patient-centered outcome variables after immediate or delayed loading.

Association of skirt size and postmenopausal breast cancer risk in older women: a cohort study within the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).

OBJECTIVES: Several studies suggest that overall and central-obesity are associated with increased breast cancer (BC) risk in postmenopausal-women. However, there are no studies investigating changes of central obesity and BC. We report on the association of BC risk with self-reported skirt size (SS; waist-circumference proxy) changes between 20s and postmenopausal-age. DESIGN: Prospective cohort-study. SETTING: UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) involving the nine trial centres in England. PARTICIPANTS: Postmenopausal-women aged >50 with no known history of BC prior to or on the day of completion of the study-entry questionnaire. INTERVENTIONS: At recruitment and at study entry, women were asked to complete a questionnaire. Women were followed-up via 'flagging' at the NHS Information Centre in England and the Hospital Episode Statistics. MAIN OUTCOME-MEASURE: Time to initial BC diagnosis. RESULTS: Between 2 January 2005 and 1 July 2010, 92,834 UKCTOCS participants (median age 64.0) completed the study-entry questionnaire. During median follow-up of 3.19 years (25th-75th centile: 2.46-3.78), 1090 women developed BC. Model adjusted analysis for potential confounders showed body mass index (BMI) at recruitment to UKCTOCS (HR for a 5 unit change=1.076, 95% CI 1.012 to 1.136), current SS at study entry (HR=1.051; 95% CI 1.014 to 1.089) and change in SS per 10 years (CSS) (HR=1.330; 95% CI 1.121 to 1.579) were associated with increased BC risk but not SS at 25 (HR=1.006; 95% CI 0.958 to 1.056). CSS was the most predictive singe adiposity measure and further analysis including both CSS and BMI in the model revealed CSS remained significant (HR=1.266; 95% CI 1.041 to 1.538) but not BMI (HR=1.037; 95% CI 0.970 to 1.109). CONCLUSIONS: CSS is associated with BC risk independent of BMI. A unit increase in UK SS (eg, 12-14) every 10-years between 25 and postmenopausal-age is associated with postmenopausal BC risk by 33%. Validation of these results could provide women with a simple and easy to understand message. TRIAL REGISTRATION NUMBER: ISRCTN22488978.

A randomized clinical trial comparing guided implant surgery (bone- or mucosa-supported) with mental navigation or the use of a pilot-drill template.

AIM: To assess the accuracy of guided surgery (mucosa and bone-supported) compared to mental navigation or the use of a surgical template, in fully edentulous jaws, in a randomized controlled study. MATERIAL AND METHODS: Fifty-nine patients (72 jaws), requiring four to six implants (maxilla or mandible), were consecutively recruited and randomly assigned to one of the following treatment groups; guidance via Materialise Universal(®)/mucosa, Materialise Universal(®)/bone, Facilitate™/mucosa, Facilitate™/bone, or mental navigation or a pilot-drill template. The precision was assessed by matching the planning computed tomography (CT) with a post-operative cone beam CT. RESULTS: A significant lower mean deviation at the entry point (1.4 mm, range: 0.3-3.7), at the apex (1.6 mm, range: 0.2-3.7) and angular deviation (3.0°, range: 0.2-16°) was observed for the guiding systems when compared to mental navigation (2.7 mm, range: 0.3-8.3; 2.9 mm, range: 0.5-7.4 and 9.9°, range: 1.5-27.8) and to the surgical template group (3.0 mm, range: 0.6-6.6; 3.4 mm, range: 0.3-7.5 and 8.4°, range: 0.6-21.3°). Differences between bone and mucosa support or type of guidance were negligible. Jaw and implant location (posterior-anterior, left-right), however, had a significant influence on the accuracy when guided. CONCLUSION: Based on these findings, guided implant placement appears to offer clear accuracy benefits.

Angiosarcoma presenting with minor erythema and swelling.

A 76-year-old man presented with slowly progressive swelling in his forehead and left upper eyelid over the course of three months. CT scanning showed non-specific enhancement of subcutaneous tissues, suggesting a low-grade cellulitis. Poor response to treatment prompted an MRI, which revealed the presence of a soft tissue lesion. Biopsy of this lesion was positive for angiosarcoma. The patient underwent chemotherapy and radiation, but unfortunately succumbed to his malignancy eight months later. This case illustrates a rare example of facial/periorbital angiosarcoma, a benign-appearing but aggressive tumor associated with a high incidence of mortality. A review of the literature and current treatment options are discussed.

Unlike Th1, Th17 cells mediate sustained autoimmune inflammation and are highly resistant to restimulation-induced cell death.

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4(+) cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.

Both Th1 and Th17 are immunopathogenic but differ in other key biological activities.

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.

A meta-analysis of risk factors that predict psychopathology following accidental trauma.

PURPOSE: This meta-analysis aimed to explore the risk factors that place a child at risk of psychopathology following accidental trauma. DESIGN AND METHODS: The predictive power of 8 factors was examined via transforming and combining the effect sizes to yield a weighted average effect size for each factor. RESULTS: The results indicated that the majority of effect sizes, although significant, were inconsistent across the studies, yielding little conclusive evidence. However, pretrauma psychopathology and threat to life were strong and consistent predictors. PRACTICE IMPLICATIONS: Information gathered from such meta-analyses could be used in the identification of at-risk children and the development of screening tools. However, further widespread and comprehensive reviews of the potential risk factors and their relationships to psychopathology need to be investigated.

Intimate partner violence: implications for critical care nursing.

IPV presents a serious health risk to many women. Emergency and critical care nurses are in a prime position for identification of and intervention with these women. Careful assessment and recognition of symptoms and conditions associated with IPV helps nurses to identify victims and potential victims. Building a trusting and supportive environment, where women feel comfortable disclosing abuse, precedes effective intervention. Nursing practice can incorporate the myriad resources and successful programs to provide victims with quality care. Before discharge from the critical care unit or ED, nurses can help women to identify their risk and plan for safety. Effective nursing care in IPV combines the traditional critical care nursing skills synergistically with provision for the biopsychosocial needs of patients.

Optimization of methods to assess human mucosal T-cell responses to HIV infection.

The majority of HIV-1 infections occur via sexual transmission at mucosal epithelia lining the vagina, cervix or rectum. Mucosal tissues also serve as viral reservoirs. However, our knowledge of human mucosal T-cell responses is limited. There is a need for reliable, sensitive, and reproducible methods for assessing mucosal immunity. Here we report on the collaborative efforts of two laboratories to optimize methods for processing, culturing, and analyzing mucosal lymphocytes. Rectal biopsy tissue was obtained by flexible sigmoidoscopy, which is rapid, minimally invasive, and well tolerated. Of the four methods compared for isolating mucosal mononuclear cells (MMC), collagenase digestion reproducibly yielded the most lymphocytes (4-7 x 10(6)). Furthermore, 0.5-1 x 10(6) MMC could be polyclonally expanded to yield 17 x 10(6) CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-gamma enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-gamma production normally detected in fresh MMC. Piperacillin-tazobactam and amphotericin B reduced contamination of MMC cultures to 4%. Taken together, these methods will be useful for studies of mucosal immunity to HIV-1 and other pathogens during natural infection and following vaccination.

Trafficking of human immunodeficiency virus type 1-specific CD8+ T cells to gut-associated lymphoid tissue during chronic infection.

Gut-associated lymphoid tissue (GALT) is a significant but understudied lymphoid organ, harboring a majority of the body's total lymphocyte population. GALT is also an important portal of entry for human immunodeficiency virus (HIV), a major site of viral replication and CD4(+) T-cell depletion, and a frequent site of AIDS-related opportunistic infections and neoplasms. However, little is known about HIV-specific cell-mediated immune responses in GALT. Using lymphocytes isolated from rectal biopsies, we have determined the frequency and phenotype of HIV-specific CD8(+) T cells in human GALT. GALT CD8(+) T cells were predominantly CD45RO(+) and expressed CXCR4 and CCR5. In 10 clinically stable, chronically infected individuals, the frequency of HIV Gag (SL9)-specific CD8(+) T cells was increased in GALT relative to peripheral blood mononuclear cells by up to 4.6-fold, while that of cytomegalovirus (CMV)-specific CD8(+) T cells was significantly reduced (P = 0.012). Both HIV- and CMV-specific CD8(+) T cells in GALT expressed CCR5, but only HIV-specific CD8(+) T cells expressed alpha E beta 7 integrin, suggesting that mucosal priming may account for their retention in GALT. Chronically infected individuals exhibited striking depletion of GALT CD4(+) T cells expressing CXCR4, CCR5, and alpha E beta 7 integrin, but CD4(+)/CD8(+) T-cell ratios in blood and GALT were similar. The percentage of GALT CD8(+) T cells expressing alpha E beta 7 was significantly decreased in infected individuals, suggesting that HIV infection may perturb lymphocyte retention in GALT. These studies demonstrate the feasibility of using tetramers to assess HIV-specific T cells in GALT and reveal that GALT is the site of an active CD8(+) T-cell response during chronic infection.

Live, attenuated simian immunodeficiency virus SIVmac-M4, with point mutations in the Env transmembrane protein intracytoplasmic domain, provides partial protection from mucosal challenge with pathogenic SIVmac251.

Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain. This domain has been implicated in viral assembly, infectivity, and cytopathogenicity. Whereas parental SIVmac239-Nef(+) induced persistent viremia and simian AIDS in rhesus macaques, SIVmac-M4 induced transient viremia in juvenile and neonatal macaques, with no disease for at least 1 year postinfection. In this vaccine study, 8 macaques that were infected as juveniles (n = 4) or neonates (n = 4) with SIVmac-M4 were challenged with pathogenic SIVmac251 administered through oral mucosa. At 1 year postchallenge, six of the eight macaques had low to undetectable plasma viremia levels. Assays of cell-mediated immune responses to SIVmac Gag, Pol, Env, and Nef revealed that all animals developed strong CD8(+) T-cell responses to Gag after challenge but not before. Unvaccinated control animals challenged with SIVmac251 developed persistent viremia, had significantly weaker SIV-specific T-cell responses, and developed AIDS-related symptoms. These findings demonstrate that SIVmac-M4, which contains a full-length Nef coding region and multiple point mutations in the TM, can provide substantial protection from mucosal challenge with pathogenic SIVmac251.

Cellular longevity: role of apoptosis and replicative senescence.

Cellular longevity refers to the lifespan of an individual cell. Normal cells have a finite lifespan and typically die by undergoing apoptosis, or enter into a state of irreversible growth arrest, termed replicative senescence, at the end of that lifespan. The lifespan of a cell is a balance between pro-survival/anti-apoptotic and pro-apoptotic death-promoting factors. The role of heat shock proteins, Bcl-2 family members, antioxidant molecules, and telomere length and telomerase activity in the regulation of apoptosis and replicative senescence, will be discussed.