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Objective: A study was undertaken to characterise how doctors in emergency medicine (EM), acute medicine (AM) and critical care (ICU) in the UK, USA and Australia and New Zealand (ANZ) approach the initial resuscitative care of patients with severe sepsis. Methods: In 2007, members on the mailing lists of UK, US and ANZ EM, ICU and AM specialist organisations were invited to answer an anonymous scenario-based online survey. Respondents described their management of a patient with pneumonia and signs of sepsis. Multiple-choice questions were based on the Surviving Sepsis Campaign (SSC) 6-hour resuscitation bundle guidelines while avoiding the specific terms "sepsis" and "SSC guidelines". Results: The response rate was 21% (2461/11 795). Only two respondents (0.1%) complied with all SSC resuscitation recommendations. Inter-specialty and inter-country variations included differences in reporting initial lactate measurement (ranging from 30% in US-EM to 79% in UK-EM), fluid resuscitation targeting a central venous pressure of 8-12 mm Hg (from 15% in ANZ-ICU to 60% in UK-EM), blood transfusion for a central venous oxygen saturation <70% and haematocrit <30% (from 15% in ANZ-ICU to 70% in US-EM and UK-EM) and insertion of invasive monitoring (intra-arterial catheter: 89% in UK-ICU vs 20% in US-EM; central venous catheter: 83% in UK-ICU vs 44% in US-EM). 81% of respondents identified at least one reason why they did not implement all the recommendations; the reasons varied by region and specialty. Conclusions: Reported management of early sepsis varies between specialities and countries, and the responses do not follow SSC guidelines. Concerns relate to knowledge, attitudes and resources. [ABSTRACT FROM AUTHOR]

Individual differences in reading ability are associated with characteristics of white matter microstructure in the brain. However, previous studies have largely measured reading as a single construct, resulting in difficulty characterizing the role of structural connectivity in discrete subskills of reading. The present study used diffusion tensor imaging to examine how white matter microstructure, measured by fractional anisotropy (FA), relates to individual differences in reading subskills in children aged 8 to 14 (n = 65). Findings showed positive correlations between FA of the left arcuate fasciculus and measures of single word reading and rapid naming abilities. Negative correlations were observed between FA of the right inferior longitudinal fasciculus and bilateral uncinate fasciculi, and reading subskills, particularly reading comprehension. The results suggest that although reading subskills rely to some extent on shared tracts, there are also distinct characteristics of white matter microstructure supporting different components of reading ability in children., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Individual differences in reading ability have been linked to characteristics of functional connectivity in the brain in both children and adults. However, many previous studies have used single or composite measures of reading, leading to difficulty characterizing the role of functional connectivity in discrete subskills of reading. The present study addresses this issue using resting-state fMRI to examine how resting-state functional connectivity (RSFC) related to individual differences in children's reading subskills, including decoding, sight word reading, reading comprehension, and rapid automatized naming (RAN). Findings showed both positive and negative RSFC-behaviour relationships that diverged across different reading subskills. Positive relationships included increasing RSFC among left dorsal and anterior regions with increasing decoding proficiency, and increasing RSFC between the left thalamus and right fusiform gyrus with increasing sight word reading, RAN, and reading comprehension abilities. In contrast, negative relationships suggested greater functional segregation of attentional and reading networks with improved performance on RAN, decoding, and reading comprehension tasks. Importantly, the results suggest that although reading subskills rely to some extent on shared functional networks, there are also distinct functional connections supporting different components of reading ability in children., (Copyright © 2021. Published by Elsevier Inc.)

Triple-Negative Breast Cancers (TNBCs) constitute roughly 10-20% of breast cancers and are associated with poor clinical outcomes. Previous work from our laboratory and others has determined that the cytoplasmic adaptor protein Breast Cancer Antiestrogen Resistance 3 (BCAR3) is an important promoter of cell motility and invasion of breast cancer cells. In this study, we use both in vivo and in vitro approaches to extend our understanding of BCAR3 function in TNBC. We show that BCAR3 is upregulated in ductal carcinoma in situ (DCIS) and invasive carcinomas compared to normal mammary tissue, and that survival of TNBC patients whose tumors contained elevated BCAR3 mRNA is reduced relative to individuals whose tumors had less BCAR3 mRNA. Using mouse orthotopic tumor models, we further show that BCAR3 is required for efficient TNBC tumor growth. Analysis of publicly available RNA expression databases revealed that MET receptor signaling is strongly correlated with BCAR3 mRNA expression. A functional role for BCAR3-MET coupling is supported by data showing that both proteins participate in a single pathway to control proliferation and migration of TNBC cells. Interestingly, the mechanism through which this functional interaction operates appears to differ in different genetic backgrounds of TNBC, stemming in one case from potential differences in the strength of downstream signaling by the MET receptor and in another from BCAR3-dependent activation of an autocrine loop involving the production of HGF mRNA. Together, these data open the possibility for new approaches to personalized therapy for individuals with TNBCs., Competing Interests: None., (AJCR Copyright © 2021.)

The present work considers how connectome-wide differences in brain organization might distinguish good and poor readers. The connectome comprises a 'rich-club' organization in which a small number of hub regions play a focal role in assisting global communication across the whole brain. Prior work indicates that this rich-club structure is associated with typical and impaired cognitive function although no work so far has examined how this relates to skilled reading or its disorders. Here we investigated the rich-club structure of brain's white matter connectome and its relationship to reading subskills in 64 children with and without reading disabilities. Among three types of white matter connections, the strength of feeder connections that connect hub and non-hub nodes was significantly correlated with word reading efficiency and phonemic decoding. Phonemic decoding was also positively correlated with connectivity between connectome-wide hubs and nodes within the left-hemisphere reading network, as well as the local efficiency of the reading network. Exploratory analyses also identified sex differences indicating these effects were stronger in girls. This work highlights the independent roles of connectome-wide structure and the more narrowly-defined reading network in understanding the neural bases of skilled and impaired reading in children., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: In 2012, the Centers for Disease Control and Prevention (CDC) Advisory Council on Immunization Practice recommended an additional post-splenectomy booster vaccine at 8 weeks following the initial vaccine. The objective of this study was to evaluate our vaccination compliance rate and what sociodemographic factors were associated with noncompliance following this recommendation., Materials and Methods: A retrospective review of a performance improvement database of trauma patients eligible for post-splenectomy vaccination (PSV) at a level I trauma center was carried out between 2009 and 2018. Overall and institutional compliance with PSV was compared before and after the addition of booster vaccine recommendation. Factors associated with booster noncompliance were also identified., Results: A total of 257 patients were identified. PSV compliance rate in the pre-booster was 98.4%, while overall and institutional post-booster compliance rate were significantly lower at 66.9% ( P ≤ .001) and 50.0% ( P ≤ .001), respectively. Compared to booster institutional compliers, institutional noncompliers lived farther from the trauma center (48 vs. 86 miles, P = .02), and though not statistically significant, these patients were generally older (34.9 vs. 40.5, P = .05)., Discussion: PSV booster compliance is low even with the current educational materials and recommendations. Additional approaches to improve compliance rates need to be implemented, such as sending letters to the patient and their primary care providers (PCPs), collaborating with rehab/long-term acute care centers, communicating with city and county health departments and city pharmacies, or mirroring other countries and creating a national database for asplenic patients to provide complete information.

Background: Retained rectal foreign bodies are a common but incompletely studied problem. This study defined the epidemiology, injury severity, and outcomes after rectal injuries following foreign body insertion., Methods: Twenty-two level I trauma centers retrospectively identified all patients sustaining a rectal injury in this AAST multi-institutional trial (2005-2014). Only patients injured by foreign body insertion were included in this secondary analysis. Exclusion criteria were death before rectal injury management or ≤48 h of admission. Demographics, clinical data, and outcomes were collected. Study groups were defined as partial thickness (AAST grade I) versus full thickness (AAST grades II-V) injuries. Subgroup analysis was performed by management strategy (nonoperative versus operative)., Results: After exclusions, 33 patients were identified. Mean age was 41 y (range 18-57), and 85% (n = 28) were male. Eleven (33%) had full thickness injuries and 22 (67%) had partial thickness injuries, of which 14 (64%) were managed nonoperatively and 8 (36%) operatively (proximal diversion alone [n = 3, 14%]; direct repair with proximal diversion [n = 2, 9%]; laparotomy without rectal intervention [n = 2, 9%]; and direct repair alone [n = 1, 5%]). Subgroup analysis of outcomes after partial thickness injury demonstrated significantly shorter hospital length of stay (2 ± 1; 2 [1-5] versus 5 ± 2; 4 [2-8] d, P = 0.0001) after nonoperative versus operative management., Conclusions: Although partial thickness rectal injuries do not require intervention, difficulty excluding full thickness injuries led some surgeons in this series to manage partial thickness injuries operatively. This was associated with significantly longer hospital length of stay. Therefore, we recommend nonoperative management after a retained rectal foreign body unless full thickness injury is conclusively identified., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS - FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS - FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1 We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Purpose This review article provides a scoping review of the literature on mathematical abilities in developmental language disorder (DLD). Children with DLD typically struggle with learning in school; however, the mechanism by which DLD impacts academic success is unclear. Mathematics involves demands in the multiple domains and therefore holds potential for examining the relationship between language and academic performance on tasks mediated by verbal and nonverbal demands. Method A scoping review was performed via computerized database searching to examine literature on mathematics and DLD. The 21 review articles meeting inclusion criteria compared children with typical development or DLD on various tasks measuring numerical cognition. Results Children with DLD consistently performed below peers with typical development on number transcoding, counting, arithmetic, and story problem tasks. However, performance was similar to peers with typical development on most number line, magnitude comparison, and conceptual mathematics tasks. Conclusions The findings suggest a relationship between DLD and mathematics was characterized by more detrimental performance on tasks with higher verbal demands. Results are discussed with respect to typical academic curricula and demonstrate a need for early identification and intervention in DLD to optimize academic outcomes.

Background: There are no clear guidelines for the best test or combination of tests to identify traumatic rectal injuries. We hypothesize that computed tomography (CT) and rigid proctoscopy (RP) will identify all injuries., Methods: American Association for the Surgery of Trauma multi-institutional retrospective study (2004-2015) of patients who sustained a traumatic rectal injury. Patients with known rectal injuries who underwent both CT and RP as part of their diagnostic workup were included. Only patients with full thickness injuries (American Association for the Surgery of Trauma grade II-V) were included. Computed tomography findings of rectal injury, perirectal stranding, or rectal wall thickening and RP findings of blood, mucosal abnormalities, or laceration were considered positive., Results: One hundred six patients were identified. Mean age was 32 years, 85(79%) were male, and 67(63%) involved penetrating mechanisms. A total of 36 (34%) and 100 (94%) patients had positive CT and RP findings, respectively. Only 3 (3%) patients had both a negative CT and negative RP. On further review, each of these three patients had intraperitoneal injuries and had indirect evidence of rectal injury on CT scan including pneumoperitoneum or sacral fracture., Conclusion: As stand-alone tests, neither CT nor RP can adequately identify traumatic rectal injuries. However, the combination of both test demonstrates a sensitivity of 97%. Intraperitoneal injuries may be missed by both CT and RP, so patients with a high index of suspicion and/or indirect evidence of rectal injury on CT scan may necessitate laparotomy for definitive diagnosis., Level of Evidence: Diagnostic, level IV.

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type., (© 2018 The Authors. Molecular Carcinogenesis Published by WileyPeriodicals, Inc.)

Introduction: Rectal injuries have been historically treated with a combination of modalities including direct repair, resection, proximal diversion, presacral drainage, and distal rectal washout. We hypothesized that intraperitoneal rectal injuries may be selectively managed without diversion and the addition of distal rectal washout and presacral drainage in the management of extraperitoneal injuries are not beneficial., Methods: This is an American Association for the Surgery of Trauma multi-institutional retrospective study from 2004 to 2015 of all patients who sustained a traumatic rectal injury and were admitted to one of the 22 participating centers. Demographics, mechanism, location and grade of injury, and management of rectal injury were collected. The primary outcome was abdominal complications (abdominal abscess, pelvic abscess, and fascial dehiscence)., Results: After exclusions, there were 785 patients in the cohort. Rectal injuries were intraperitoneal in 32%, extraperitoneal in 58%, both in 9%, and not documented in 1%. Rectal injury severity included the following grades I, 28%; II, 41%; III, 13%; IV, 12%; and V, 5%. Patients with intraperitoneal injury managed with a proximal diversion developed more abdominal complications (22% vs 10%, p = 0.003). Among patients with extraperitoneal injuries, there were more abdominal complications in patients who received proximal diversion (p = 0.0002), presacral drain (p = 0.004), or distal rectal washout (p = 0.002). After multivariate analysis, distal rectal washout [3.4 (1.4-8.5), p = 0.008] and presacral drain [2.6 (1.1-6.1), p = 0.02] were independent risk factors to develop abdominal complications., Conclusion: Most patients with intraperitoneal injuries undergo direct repair or resection as well as diversion, although diversion is not associated with improved outcomes. While 20% of patients with extraperitoneal injuries still receive a presacral drain and/or distal rectal washout, these additional maneuvers are independently associated with a three-fold increase in abdominal complications and should not be included in the treatment of extraperitoneal rectal injuries., Level of Evidence: Therapeutic study, level III.

Adhesion turnover is critical for cell motility and invasion. We previously demonstrated that the adaptor molecule breast cancer antiestrogen resistance 3 (BCAR3) promotes adhesion disassembly and breast tumor cell invasion. One of two established binding partners of BCAR3 is the adaptor molecule, p130 Cas . In this study, we sought to determine whether signaling through the BCAR3-Cas complex was responsible for the cellular functions of BCAR3. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins colocalize in dynamic cellular adhesions. Although accumulation of BCAR3 in adhesions did not require Cas binding, a direct interaction between BCAR3 and Cas was necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion molecules, α-actinin and talin, were also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex was delayed under these conditions. As was the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell chemotaxis toward serum and invasion in Matrigel. Previous work demonstrated that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. However, in contrast to wild-type BCAR3, ectopic expression of the Cas-binding mutant of BCAR3 failed to induce Rac1 activity in breast cancer cells. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell migration and invasion, and Rac1 activity is dependent on its ability to bind to Cas. The activity of BCAR3-Cas complexes as a functional unit in breast cancer is further supported by the co-expression of these molecules in multiple subtypes of human breast tumors., Competing Interests: The authors have no conflicts of interest.

There is little consensus on the definition or optimal constituents of fluid bolus therapy (FBT), and there is uncertainty regarding its physiological effects. The aims of this study were to determine clinician-reported definitions of FBT and to explore the physiological responses clinicians expect from such FBT. In June and October 2014, intensive care and emergency physicians in Australia and New Zealand were asked to participate in an electronic questionnaire of the reported practice and expectations of FBT. Two hundred and fifty-one questionnaires were completed, 65.3% from intensivists. We identified the prototypical FBT given by intensivists is more than 250 ml of compound sodium lactate, saline or 4% albumin given in less than 30 minutes, while that given by emergency department physicians is a similar volume of saline delivered over a similar time frame. Intensive care and emergency physicians expected significantly different changes in mean arterial pressure (P=0.001) and heart rate (P=0.033) following FBT. Substantial variation was demonstrated in the magnitude of expected response within both specialties for each variable. Major variations exist in self-reported FBT practice, both within and between acute specialties, and wide variation can be demonstrated in the expected physiological responses to FBT. International explorations of practice and prospective quantification of the actual physiological response to FBT are warranted.

Non-invasive ventilation refers to a number of respiratory support strategies commonly used in critical care settings. This paper describes the principles of non-invasive ventilation, the practicalities of its use and the evidence for its use in a number of common acute conditions., (© 2012 The Author; Internal Medicine Journal © 2012 Royal Australasian College of Physicians.)

In the centrosymmetric dimeric title compound, [Pr(2)(C(5)H(9)O(2))(2)(C(11)H(19)O(2))(4)(CH(3)OH)(2)], the two praseodymium(III) atoms are eight-coordinate and are bridged by O atoms from the two pivalate anions. Each Pr(III) ion is further coordinated by two chelating 2,2,6,6-tetra-methyl-3,5-hepta-nedionate (thd(-)) ligands and one methanol mol-ecule. The distance between the two Pr(III) ions is 4.273 (5) Å. Intra-molecular hydrogen bonds exists between the methanol hy-droxy group on one Pr(III) atom and a chelating O atom of a thd(-) ligand coordinated to the symmetry-related Pr(III) atom.

Reduced HLA-DR expression on monocytes has been suggested as a predictive marker of immunosuppression following very high risk surgery, but there are few reports in lower risk surgery. In 32 patients undergoing low to intermediate risk surgery, blood samples were analysed by flow cytometry for HLA-DR expression and numbers in both CD14(high) and CD14(low)CD16+ monocyte subsets. The numbers of CD14(high) monocytes increased at 24 h (mean (SD), 5.0 (2.2) vs 7.6 (3.9) x 10(5) cells.ml(-1); p < 0.01) while CD14(low)CD16+ monocytes decreased (0.68 (0.36) vs 0.44 (0.36) x 10(5) cells.ml(-1); p < 0.01). HLA-DR expression was significantly reduced in both subsets by 24 h (mean (SD) fluorescent intensity 440 (310) vs 160 (130) for CD14(high) and 1000 (410) vs 560 (380) for CD14(low)CD16+ subsets; p < 0.01). This reduction of monocyte HLA-DR expression 24 h following lower risk surgery raises questions about the purported clinical utility of this biomarker as an early predictor of postoperative complications. Our results also suggest that surgery induces significant trafficking (i.e. mobilisation, margination and extravasation) of monocyte subsets, and that monocyte HLA-DR depression is the result of a down-regulatory phenomenon (decreased protein expression on each cell) rather than the differential trafficking of monocyte subsets.

City-dwelling children from Kenya who were infected with human immunodeficiency virus type 1 (HIV-1) were tested for coinfection with cytomegalovirus (CMV), human T cell lymphotropic viruses 1 and 2, Kaposi sarcoma-associated herpesvirus (KSHV), or hepatitis B, C, and G viruses. All children were found to be coinfected with CMV, whereas 5% had hepatitis G virus coinfection and 15% had KSHV coinfection. A protective role for hepatitis G virus cannot be excluded but likely affects only a minority of HIV-1-infected African children.

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534-851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780-786 of UL9. Although the corresponding region of the homologous protein encoded by varicell-azoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.

We have developed a panel of 14 monoclonal antibodies (MAbs) to POL, the catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase encoded by gene UL30, and one MAb to the UL52 protein, another of the seven proteins essential for replication of HSV DNA. The approximate locations of the epitopes of the polymerase-specific MAbs were identified using truncated polymerase molecules, and the antibodies were characterized in a number of immunological assays allowing eight different specificities to be recognized. These MAbs, together with a polyclonal antibody raised in rabbits against a third DNA replication protein, ICP8, were used to localize the respective proteins by immunofluorescence in cells infected with wild-type HSV-1 or the DNA replication-defective mutants ambUL8 or 2-2. In BHK cells infected with ambUL8, a mutant with an amber termination codon within the coding region of gene UL8, the UL52 protein did not enter the nucleus, although ICP8 and POL entered the nucleus in a normal fashion. The failure of the UL52 protein to be correctly transported to the nucleus was also observed in both HFL and Vero cells infected with ambUL8. In contrast, UL52 protein was transported to the nucleus in BHK cells infected with wild-type HSV-1 or with 2-2, a mutant lacking a functional UL9 protein.

Measurements of oxygen deficit during submaximal exercise were correlated with the anaerobic threshold (as measured by gas exchange analysis), peak work rate on a ramp protocol, and the ability to perform constant work rate exercise in 10 male patients with New York Heart Association class 2 congestive heart failure and 12 age- and gender-matched normal controls. All subjects performed a maximal ramp exercise test for measurement of the anaerobic threshold. In addition, several 15-min constant work rate exercise sessions were conducted to evaluate oxygen deficit, measured as the area between the "ideal" square curve of oxygen consumption at the onset of constant work rate exercise and the actual exponentially shaped curve. Since the oxygen deficit significantly correlated with the plateau oxygen consumption during the 25-W constant work rate exercise (r = 0.61, p = 0.002), the oxygen deficit was normalized by the rectangular area of 15-min oxygen consumption above baseline. This normalized value significantly correlated with the inverse of the anaerobic threshold (r = 0.81, p < 0.0001). The logarithm of the normalized oxygen deficit significantly correlated with the maximum ramp work rate (r = -0.86, p < 0.0001) and the highest constant work rate sustained for 15 min (r = -0.82, p < 0.0001). In addition, the time to reach plateau oxygen consumption for the 25-W exercise significantly correlated with the inverse of the anaerobic threshold (r = -0.78, p < 0.0001), the maximum ramp work rate (r = -0.76, p < 0.0001), and the highest constant work rate sustained for 15 min (r = -0.74, p < 0.0001). Thus, the oxygen deficit seen in patients with heart failure during constant work rate exercise results from abnormally slow oxygen uptake kinetics and correlates with exercise capacity as measured by anaerobic threshold (via gas exchange analysis) and maximal and submaximal exercise tolerance. Oxygen deficit warrants further evaluation as a submaximal index of functional capacity in patients with heart failure.

The extent and time course of recovery of left ventricular function were investigated in 29 patients with no previous symptoms who had acute nonischemic congestive cardiomyopathy and left ventricular ejection fraction of 0.22 +/- 0.07. Improvement in left ventricular ejection fraction by at least 0.05 was observed in 24 of the 29 patients and was achieved within 6 months after the initial evaluation. Progressive improvement was seen, with a maximum ejection fraction of 0.45 +/- 0.17 being achieved within approximately 18 months. The degree of ejection fraction recovery was not related to the initial clinical or hemodynamic variables. However, the extent of fibrosis detected on endomyocardial biopsy correlated inversely with subsequent changes in ejection fraction (r = -0.65, p = 0.0003). Thus significant recovery is likely after an acute episode of nonischemic cardiomyopathy and may be progressive during the first year. Recovery is related to the extent of myocardial fibrosis detected on endomyocardial biopsy but cannot be predicted from the initial clinical or hemodynamic presentation.

The intracellular distributions of three herpes simplex virus type 1 (HSV-1) capsid proteins, VP23, VP5 and VP22a, were examined using vaccinia virus and plasmid expression systems. During infection of cells with HSV-1 wild-type virus, all three proteins were predominantly located in the nucleus, which is the site of capsid assembly. However, when expressed in the absence of any other HSV-1 proteins, although VP22a was found exclusively in the nucleus as expected, VP5 and VP23 were distributed throughout the cell. Thus nuclear localization is not an intrinsic property of these proteins but must be mediated by one or more HSV-1-induced proteins. Co-expression experiments demonstrated that VP5 was efficiently transported to the nucleus in the presence of VP22a, but the distribution of VP23 was unaffected by the presence of either or both of the other two proteins.

We describe a rapid method of fragment condensation to couple a helper T cell epitope, active in BALB/c mice, to the amino terminus of branched peptides (or 'multiple antigenic peptides', MAPs). The helper T cell epitope-MAP conjugate considerably enhanced the immunogenicity in BALB/c mice of branched peptides. The method of fragment condensation, whereby the helper T cell epitope portion of the immunogen is added as a 'cassette' in a one step addition process, is both faster and more convenient than continuous step-by-step addition of individual amino acids and is likely to be generally applicable. The method should be advantageous in the development of peptide based vaccines.

The herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 degrees C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt alpha-helix to a beta-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the NPT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in ts1207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface.

Immune spleen cells were treated with papain, washed, and boosted with homologous antigen (4-hydroxy-3-iodo-5-nitrophenylacetic acid coupled to chicken globulin) for 1 hr at 4 degrees C. When transferred to irradiated mice, they produced up to 20-fold less anti-hapten antibody than non-papain-treated cells. The lymphocytes could recover their ability to respond if incubated at 37 degrees C after papain treatment but before antigen boosting. Incubation for 30 min at 37 degrees C gave complete or almost complete recovery. Although papain usually reduced the response, in 31% of experiments the reduction was not significant. Possible reasons for this variability are discussed.

A hybridoma line was isolated which produced antibody reacting with polypeptides of apparent molecular weights 32,000, 34,000 and 35,000 (32K/34K/35K) and 55,000 and 57,000 (55K/57K). These were sulphated glycoproteins that have previously been found in the medium of herpes simplex virus type 1 (HSV-1)-infected cells. By tryptic peptide mapping and serological cross-reactions the polypeptides were shown to be related to HSV-1 glycoprotein E (gE-1) but they lacked the Fc binding function. The 32K/34K/35K and 55K/57K polypeptides were not found in the medium of HSV-1-infected cells incubated in serum-free medium. They could be generated in vitro from purified gE-1 in the presence of serum. It is likely that 32K/34K/35K and 55K/57K are derived from gE-1 by the action of serum proteases.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.

We report on the properties of a family of related herpes simplex virus type 1 polypeptides (designated p40) of Mr around 40,000. The intracellular localization of these polypeptides has been examined using monoclonal antibodies and their association with viral capsids within the nuclei of infected cells has been demonstrated directly by immunoelectron microscopy. Specific DNA staining and the use of mutants defective for DNA packaging has revealed, in contrast to earlier findings, that p40 is present in empty capsids. Protein p40 is not present as a major component of full capsids or of mature virions indicating that it is transiently associated with capsids and that its removal from capsids is linked with the process of DNA packaging.

Dinitrophenylated polysucrose (DNP-Ficoll) elicits T cell-independent IgM anti-DNP antibody formation in mice. This antigen also elicits a heterogeneous IgG1 and IgG2 anti-DNP response, which is operationally as T-independent as the IgM response. However, a concomitant graft-versus-host reaction markedly enhances the IgG response (allogeneic effect). These results confirm those of others, indicating that a certain proportion of the precursors of IgG-producing cells can be triggered by some T-independent antigens. However, our results suggest that even with such antigens optimal triggering of IgG precursors requires T cell help.

Experiments performed on CBA mice thymectomized in adult life, exposed to lethal doses of irradiation and given tissue therapy are described. Marrow, foetal liver, or spleen cells from syngeneic donors could protect the mice against the lethal effects of irradiation. Between 30 and 70 days' postirradiation, however, marrow-treated, thymectomized irradiated mice showed evidence of trophic disturbances, such as failure to gain weight, in contrast to sham-operated, irradiated, marrow-treated controls. The immune responses of experimental and control mice were tested up to 150 days' postirradiation by challenging with sheep erythrocytes and allogeneic skin grafts. Sham-operated irradiated controls, whether protected with marrow, foetal liver, or spleen cells, produced normal immune responses when challenged at 28, 60, or 150 days after irradiation. Neither foetal liver cells nor marrow cells, in doses of up to 40 million cells per mouse, enabled thymectomized irradiated mice to recover normal immune functions. Spleen cells, from normal donors but not from neonatally thymectomized donors, restored immunological capacity in such mice. It is concluded that immunologically competent cells are present in the spleen of normal adult donors and can function in the absence of the thymus. Bone marrow, on the other hand, does not contain an adequate population of such cells but has lymphoid precursor cells, the descendants of which can become immunologically competent only in the presence of a functioning thymus mechanism.