Helicopter winchmens' experiences with pain management in challenging environments.

We conducted a survey of Irish Coast Guard Search and Rescue Helicopter winchmen to establish if their pain management scope of practice was adequate for their working environment. We surveyed 17 SAR personnel. 88% of winchmen have experienced scenarios where they were unable to reduce pain scores below 6/10. In seeking solutions within current Irish Prehospital Clinical Practice Guidelines, repeated descriptions of operations in extreme weather and sea conditions were given which were entirely incompatible with the dexterity required to break a glass ampoule and draw up solution, let alone site an intravenous (IV) line or administer a drug via intramuscular (IM) injection. Irish Coast Guard Search and Rescue Helicopter winchmen encounter polytrauma patients in extreme pain in uniquely challenging environments. Novel solutions to pain management within this tightly governed system are urgently required.

Reproductive outcomes following recurrent first-trimester miscarriage: a retrospective cohort study.

STUDY QUESTION: What are the subsequent reproductive outcomes (livebirths, miscarriages or other adverse pregnancy outcomes or no further pregnancy) of women with recurrent miscarriage (RM) attending a dedicated clinic? SUMMARY ANSWER: Of women with RM, 77% had a subsequent pregnancy, and among these pregnancies, the livebirth rate was 63%. WHAT IS KNOWN ALREADY: RM affects ∼1-3% of women of reproductive age. RM has known associations with advanced maternal age, obesity, diabetes, inherited thrombophilias, thyroid dysfunction, endometriosis and parental balanced translocations. However, ∼ 50% of women or couples will be left without an explanation for their pregnancy loss, even after completing investigations. RM is also associated with secondary infertility and adverse pregnancy outcomes including preterm birth and perinatal death. STUDY DESIGN SIZE DURATION: We undertook a retrospective cohort study to identify subsequent pregnancy outcomes in women with RM, defined as three consecutive first-trimester miscarriages. Women attending the RM clinic at a tertiary university hospital in the Republic of Ireland over 12 years (2008-2020) with a confirmed diagnosis of primary or secondary first-trimester RM were eligible for inclusion. In total, 923 charts were identified for review against the eligibility criteria. PARTICIPANTS/MATERIALS SETTING METHODS: Women with non-consecutive first-trimester miscarriages or ectopic pregnancy were excluded. Epidemiological and clinical information regarding medical history, investigation and management was gathered from paper and electronic medical records. Data were analysed using SPSS (Version 27). Associations between maternal characteristics and outcomes were explored using the χ2 test, with significance set at P < 0.05. Multinomial regression analysis was performed using a stepwise approach. MAIN RESULTS AND THE ROLE OF CHANCE: There were 748 women who were included; 332 (44%) had primary RM and 416 (56%) had secondary RM. The median age was 36 years (range 19-47). Foetal aneuploidy was the most common investigative finding (15%; n = 111/748); 60% had unexplained RM. In addition to supportive care, most women were prescribed aspirin (96%) and folic acid (75%). Of the 748 women, 573 had a subsequent pregnancy (77%) and 359 (48% of all women; 63% of pregnancies) had a livebirth, while 208 had a further pregnancy loss (28% of all women; 36% of pregnancies) and 6 were still pregnant at the end of the study. Women aged 35-39 years were more likely to have a livebirth than no further pregnancy (relative risk ratio (RRR): 2.29 (95% CI: 1.51-5.30)). Women aged 30-34 years were more likely to have a livebirth (RRR: 3.74 (95% CI: 1.80-7.79)) or a miscarriage (RRR: 2.32 (95% CI: 1.07-4.96)) than no further pregnancy. Smokers were less likely to have a livebirth (RRR: 0.37 (95% CI: 0.20-0.69)) or a miscarriage (RRR: 0.45 (95% CI: 0.22-0.90)) than no further pregnancy. Couples with an abnormal parental karyotype were less likely to have a miscarriage than no further pregnancy (RRR: 0.09 (95% CI: 0.01-0.79)). Including successive pregnancies conceived over the study period, the overall livebirth rate was 63% (n = 466/742), but this was reduced to 44% in women aged ≥40 years and 54% in women with infertility. LIMITATIONS REASONS FOR CAUTION: This work covers 13 years; however, those included in the later years have a shorter follow-up time. Although electronic health records have improved data availability, data collection in this cohort remains hampered by the absence of a formal booking visit for women presenting with miscarriage and a national miscarriage database or register. WIDER IMPLICATIONS OF THE FINDINGS: Our findings are largely reassuring as most women with RM and hoping to conceive achieved a livebirth. In addition to older age, smoking and parental balanced translocations were associated with a reduced likelihood of further pregnancy. No investigation or treatment was associated with pregnancy outcome, reiterating the importance of the supportive aspects of care for women and their partners after RM and counselling regarding individual risk factors. This contributes to the limited international data on the investigative findings and treatment of women with RM. The high rate of prescribed medications merits greater scrutiny, in conjunction with other pregnancy outcomes, and reiterates the need for a national guideline on RM. STUDY FUNDING/COMPETING INTERESTS: L.A.L. is a PhD scholar funded through the Pregnancy Loss Research Group, Department of Obstetrics and Gynaecology, University College Cork. M.H. and C.F. are Postdoctoral Researchers on a project funded by the Health Research Board Ireland [ILP-HSR-2019-011] and led by K.O.D., titled: 'Study of the impact of dedicated recurrent miscarriage clinics in the Republic of Ireland'. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors have no conflicts of interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Regulation of the germline immunoglobulin Cgamma1 promoter by CD40 ligand and IL-4: dual role for tandem NF-kappaB binding sites.

Transcription of germline Ig constant region genes and associated switch regions is an early and essential step in heavy chain class switch recombination. Transcription of the germline Cgamma1 and C epsilon Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcription of these genes and act in synergy with IL-4 to increase expression. In the present study, we investigated the role of three tandem NF-kappaB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cgamma1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL1-3B3. Germline gamma1 transcripts are induced by CD40L and by IL-4 in BCL1-3B3 and the combination of signals is synergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulation with CD40L results in the induction of NF-kappaB complexes that bind to each of the three NF-kappaB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-kappaB sites did not reduce CD40-responsiveness of germline gamma1 promoter-luciferase reporter constructs transiently transfected into BCL1-3B3. Mutation in any one NF-kappaB site, however, significantly reduced overall transcriptional activity of the promoter, both basal and induced, suggesting a role in basal promoter function. In addition, activation of the promoter by IL-4 was blocked by mutation of all three NF-kappaB sites and similarly reduced by mutation of site 1, suggesting that NF-kappaB-STAT6 interactions may be necessary for STAT6-mediated transactivation of the germline gamma1 promoter. The results suggest that the three NF-kappaB sites may serve as a focus for formation of a higher-order transcription complex including STAT6, NF-kappaB and components of the basal transcription apparatus.

The antineoplastic effects of tuftsin and tuftsinyltuftsin on B16/5B melanoma and L1210 cells.

The intraperitoneal (i.p.) injection of tuftsin, Thr-Lys-Pro-Arg, into C57BL/6 mice that were injected with B16/5B melanoma cells, resulted in a considerable suppression and elimination of solid tumor growth. While 100% of control animals exhibited tumor growth, 38% of the treated animals failed to show tumor formation for the duration of the experiment, 60-80 days. The octapeptide, tuftsinyltuftsin, was effective at 3 ng per mouse as was a dose of 2 and 20 micrograms per mouse. In each case there was a significant number of mice free of tumors. The octapeptide was also quite effective against L1210 cells resulting in the survival of 35-40% of the treated animals. The lethal effect of increased superoxide, O X 2, production by tuftsin treatment may explain the antineoplastic effect of the tetrapeptide. This may result not only from higher concentrations of O X 2 but also from the potentially lethal effects of H2O2 and OH X radical, both of which are products of O X 2 metabolism.

Extrahepatic expression of plasma protein genes during inflammation.

The body's protective responses to infection, wounding, trauma, and malignancy include the acute-phase reaction, which is modulated by various cytokines and their cellular receptors. During the acute-phase reaction, levels of specific proteins synthesized by the liver increase in the plasma. Little information is available about the extrahepatic synthesis of plasma proteins during the acute-phase reaction. The study described here analyzes the tissue-specific expression of genes encoding the plasma proteins albumin (ALB), alpha 1-antitrypsin (AAT), transferrin (TF), haptoglobin (HP), ceruloplasmin (CP), serum amyloid A (SAA), alpha 1-acid glycoprotein (AGP) and alpha 2-HS-glycoprotein (AHSG) during the acute-phase reaction in C57B1 mice. The acute-phase reaction was induced by intraperitoneal injections of bacterial lipopolysaccharide (LPS). During the acute-phase reaction, genes encoding CP, SAA, AGP, and HP demonstrate unique extrahepatic tissue specific patterns of expression in kidney, spleen, thymus, heart, brain, lung, testis, and epididymis. Different temporal patterns of HP gene expression also were observed in lung and thymus after induction by LPS. The function of extrahepatic synthesis of plasma proteins is not yet understood; however, a local provision of specific plasma proteins in mammalian tissues may offer the host a source of functionally important proteins during periods of stress.

Candida albicans C3d receptor, isolated by using a monoclonal antibody.

Pseudohyphae of Candida albicans possess a receptor for C3d, a fragment of the complement component C3. This receptor was partially purified by using a monoclonal antibody (CA-A) that previously had been shown to inhibit the binding of C3d to C. albicans pseudohyphae. Purified immunoglobulin G from ascites fluid (CA-A) was coupled to a cyanogen bromide-activated Sepharose column, and an affinity-purified fraction (A2) from C. albicans pseudohyphae was obtained. This fraction inhibited rosetting of the EAC3d receptor by pseudohyphae and appeared to contain glycoprotein, since receptor activity could be removed when A2 was incubated with lectins specific for mannose and glucose. A2 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two polypeptides of approximately 60 and 70 kilodaltons (kDa) were consistently identified in reducing gels. The 60-kDa protein was identified as a glycoprotein by concanavalin A binding. A2 was further analyzed by high-pressure liquid chromatography (HPLC). Of three fractions obtained by HPLC, one containing the 60-kDa protein was found to have receptor activity. When analyzed by HPLC, this protein was found to contain mannose and glucose in approximately equal amounts. Both immunofluorescence and electron microscopy of pseudohyphae treated with CA-A identified A2 as a surface moiety. Thus, the C3d receptor of C. albicans, isolated with CA-A, is a glycoprotein of approximately 60 kDa.

IL-4 activates a latent DNA-binding factor that binds a shared IFN-gamma and IL-4 response element present in the germ-line gamma 1 Ig promoter.

IL-4 regulates transcription of the germ-line gamma 1 Ig gene in murine B cells and by doing so targets this isotype for switch recombination by an unknown mechanism. In this study, we have identified an IL-4-induced DNA-binding protein factor in murine B cells designated NF-IL-4-gamma 1. This factor binds specifically to a site within a 13-bp DNA sequence extending from -125 to -113 (5' CATTCACATGAAG 3') in the germ-line gamma 1 promoter and shown previously to be important for IL-4-responsive transcription. This sequence is highly homologous to the IFN-gamma activation site or GAS, and competitive binding studies demonstrate that NF-IL-4-gamma 1 can also bind to GAS elements in the promoters of two IFN-gamma-responsive genes and to an IL-4-responsive element in the germ-line epsilon Ig promoter. NF-IL-4-gamma 1 is rapidly induced in the absence of de novo protein synthesis and expression is sustained through day 4 of in vitro culture with IL-4 and LPS. Induction of NF-IL-4-gamma 1 is inhibited by the kinase inhibitor staurosporine and the factor itself requires phosphorylation for binding activity. The binding specificity and expression characteristics of NF-IL-4-gamma 1 suggest identity with other recently described IL-4-activated, GAS-binding factors that are members of the signal transducers and activators of transcription (STAT) family of cytokine-responsive transcription factors.

Identification of C3d receptors on Candida albicans.

Pseudohyphal but not yeast forms of Candida albicans possess both iC3b and C3d receptors, as determined by rosetting with erythrocytes carrying iC3b (EAC3bi) or C3d (EAC3d). Rosetting with EAC3d was markedly reduced when pseudohyphae were heat killed or treated with trypsin or pronase but was not inhibited by several saccharides or aminosaccharides, including alpha-methyl-D-mannoside, or by pretreatment of pseudohyphae with concanavalin A. However, mannoproteins obtained by concanavalin A affinity chromatography of whole pseudohyphal extracts inhibited the attachment of EAC3d to C. albicans, whereas soluble (nonmannosylated) proteins were less active. Thus, although the C3d receptors appeared to be glycosylated, the oligosaccharide component of the receptor was apparently not involved in the recognition of C3d. To isolate these receptors, whole-cell extracts were separated by DEAE-Trisacryl chromatography. Fractions that inhibited rosetting were pooled and affinity purified by C3d-Thiol-Sepharose chromatography. The eluate from this affinity column inhibited attachment of C. albicans to EAC3d. Monoclonal antibodies to C. albicans were prepared, and three of these antibodies blocked rosetting. Western blotting (immunoblotting) with these antibodies indicated the presence of 62- and 70-kilodalton receptors for C3d in the extracts purified by C3d affinity chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Tuftsin, a natural activator of phagocytic functions including tumoricidal activity.

Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in prolonging the life of syngeneic mice injected with L1210 leukemia cells. Its effect in our laboratory, was considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high performance liquid chromatography is described using 0.75% trifluoroacetic acid in water. The tuftsin sequence Thr-Lys-Pro-Arg is present in P12 protein of Rausher murine leukemia virus. A close analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second close analog Thr-Arg-Pro-Arg forms the penultimate carboxyterminal of a pancreatic polypeptide found in human and several animals.

Characterization of the mouse haptoglobin gene.

Plasmids containing mouse cDNA encoding haptoglobin, a major plasma protein that binds free hemoglobin, have been isolated and characterized. The amino acid sequence predicted by the mouse haptoglobin cDNA was 80% identical to human haptoglobin and 90% identical to rat haptoglobin sequence. The mouse haptoglobin probe was used to demonstrate a single haptoglobin gene in the genome of C57BL6 mice mapped to chromosome 8. Sequence analysis of the mouse Hp gene promoter revealed two unique features: the presence of a second TATA box with a 48-bp trinucleotide repeat immediately upstream. The enhancer element and the sequences shown to be required for cytokine and hormonal regulation of the rat Hp gene are highly conserved in mouse. Interestingly, the single nucleotide variation G to A, which completely inactivates the IL-6 responsive element A in the rat Hp gene, is identical in mouse. This suggests that the presence of an inactive IL-6-responsive element A in Hp genes is common in rodents.

Mapping and conservation of the group-specific component gene in mouse.

The group-specific component (GC), also known as the vitamin D-binding protein, transports vitamin D and its metabolites in plasma to target tissues throughout the body. The GC gene shares an evolutionary origin with genes encoding albumin (ALB) and alpha-fetoprotein (AFP). All three genes are descendants of an evolutionary ancestor that arose from an intragenic triplication. As a result, each gene is composed of three homologous domains. The study described here characterizes and compares mouse GC to the corresponding nucleotide and amino acid sequences of GC from human and rat. The deduced amino acid sequence of mouse GC was 78% identical to human and 91% identical to rat GC. The results suggest that, unlike the corresponding sequences in the ALB and AFP genes, chromosomal sequences encoding the first domain and the leader sequence of the GC gene have specifically been conserved throughout vertebrate evolution. Protection of domain I during evolution may correlate with an important functional aspect of its sequence. The mouse GC gene was mapped to chromosome 5, where the ALB and AFP genes are also located, demonstrating conservation of the three genes in vertebrate species.

STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination.

Transcription of the germline C gamma1 and C epsilon Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline gamma1 and epsilon transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline C gamma1 and C epsilon genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline gamma1 or epsilon transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to S gamma1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline gamma1 transcript expression and switch recombination to S gamma1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells.