Omicron infection following vaccination enhances a broad spectrum of immune responses dependent on infection history.

Pronounced immune escape by the SARS-CoV-2 Omicron variant has resulted in many individuals possessing hybrid immunity, generated through a combination of vaccination and infection. Concerns have been raised that omicron breakthrough infections in triple-vaccinated individuals result in poor induction of omicron-specific immunity, and that prior SARS-CoV-2 infection is associated with immune dampening. Taking a broad and comprehensive approach, we characterize mucosal and blood immunity to spike and non-spike antigens following BA.1/BA.2 infections in triple mRNA-vaccinated individuals, with and without prior SARS-CoV-2 infection. We find that most individuals increase BA.1/BA.2/BA.5-specific neutralizing antibodies following infection, but confirm that the magnitude of increase and post-omicron titres are higher in the infection-naive. In contrast, significant increases in nasal responses, including neutralizing activity against BA.5 spike, are seen regardless of infection history. Spike-specific T cells increase only in infection-naive vaccinees; however, post-omicron T cell responses are significantly higher in the previously-infected, who display a maximally induced response with a highly cytotoxic CD8+ phenotype following their 3rd mRNA vaccine dose. Responses to non-spike antigens increase significantly regardless of prior infection status. These findings suggest that hybrid immunity induced by omicron breakthrough infections is characterized by significant immune enhancement that can help protect against future omicron variants.

Longitudinal Lung Function Assessment of Patients Hospitalized With COVID-19 Using 1H and 129Xe Lung MRI.

BACKGROUND: Microvascular abnormalities and impaired gas transfer have been observed in patients with COVID-19. The progression of pulmonary changes in these patients remains unclear. RESEARCH QUESTION: Do patients hospitalized with COVID-19 without evidence of architectural distortion on structural imaging exhibit longitudinal improvements in lung function measured by using 1H and 129Xe MRI between 6 and 52 weeks following hospitalization? STUDY DESIGN AND METHODS: Patients who were hospitalized with COVID-19 pneumonia underwent a pulmonary 1H and 129Xe MRI protocol at 6, 12, 25, and 51 weeks following hospital admission in a prospective cohort study between November 2020 and February 2022. The imaging protocol was as follows: 1H ultra-short echo time, contrast-enhanced lung perfusion, 129Xe ventilation, 129Xe diffusion-weighted, and 129Xe spectroscopic imaging of gas exchange. RESULTS: Nine patients were recruited (age 57 ± 14 [median ± interquartile range] years; six of nine patients were male). Patients underwent MRI at 6 (n = 9), 12 (n = 9), 25 (n = 6), and 51 (n = 8) weeks following hospital admission. Patients with signs of interstitial lung damage were excluded. At 6 weeks, patients exhibited impaired 129Xe gas transfer (RBC to membrane fraction), but lung microstructure was not increased (apparent diffusion coefficient and mean acinar airway dimensions). Minor ventilation abnormalities present in four patients were largely resolved in the 6- to 25-week period. At 12 weeks, all patients with lung perfusion data (n = 6) showed an increase in both pulmonary blood volume and flow compared with 6 weeks, although this was not statistically significant. At 12 weeks, significant improvements in 129Xe gas transfer were observed compared with 6-week examinations; however, 129Xe gas transfer remained abnormally low at weeks 12, 25, and 51. INTERPRETATION: 129Xe gas transfer was impaired up to 1 year following hospitalization in patients who were hospitalized with COVID-19 pneumonia, without evidence of architectural distortion on structural imaging, whereas lung ventilation was normal at 52 weeks.

Clinical handover communication at maternity shift changes and women's safety in Banjul, the Gambia: a mixed-methods study.

BACKGROUND: Clinical handover is a vital communication process for patient safety; transferring patient responsibility between healthcare professionals (HCPs). Exploring handover processes in maternity care is fundamental for service quality, addressing continuity of care and maternal mortality. METHODS: This mixed-methods study was conducted in all three maternity hospitals in Banjul, The Gambia. Shift-to-shift maternity handovers were observed and compared against a standard investigating content and environment. Semi-structured interviews and focus group discussions with doctors, midwives and nurses explored handover experience. RESULTS: One hundred ten nurse/midwife shift-to-shift handovers were observed across all shift times and maternity wards; only 666 of 845 women (79%) were handed over. Doctors had no scheduled handover. Shift-leads alone gave/received handover, delayed [median 35 min, IQR 24-45] 82% of the time; 96% of handovers were not confidential and 29% were disrupted. Standardised guidelines and training were lacking. A median 6 of 28 topics [IQR 5-9] were communicated per woman. Information varied significantly by time, high-risk classification and location. For women in labour, 10 [IQR 8-14] items were handed-over, 8 [IQR 5-11] for women classed 'high-risk', 5 [IQR 4-7] for ante/postnatal women (p < 0.001); > 50% had no care management plan communicated. Twenty-one interviews and two focus groups were conducted. Facilitators and barriers to effective handover surrounding three health service factors emerged; health systems (e.g. absence of formalised handover training), organisation culture (e.g. absence of multidisciplinary team handover) and individual clinician factors (e.g. practical barriers such as transportation difficulties in getting to work). CONCLUSION: Maternity handover was inconsistent, hindered by contextual barriers including lack of team communication and guidelines, delays, with some women omitted entirely. Findings alongside HCPs views demonstrate feasible opportunities for enhancing handover, thereby improving women's safety.

Fatal case of delayed-onset haemolytic anaemia after oral artemether-lumefantrine.

Artemisinin derivatives are used globally in the management of falciparum malaria. Postartemisinin delayed haemolysis (PADH) is a recognised adverse event contributing to severe anaemia. To the best of our knowledge, we report the first recorded fatal case of PADH. A 60-year-old woman presented with two episodes of collapse at home and feeling generally unwell. She had recently been treated for uncomplicated falciparum malaria 1 month prior with artemether 80 mg/lumefantrine 480 mg in Congo. Her results on admission revealed an anaemia (haemoglobin 43 g/L), raised lactate dehydrogenase and positive direct antiglobulin test that suggested an intravascular haemolytic process. She made a capacitous decision to refuse blood products in line with her personal beliefs. Despite best supportive treatment, she did not survive. This case highlights the importance of postartemisinin follow-up and should encourage discussion and careful consideration of its use in the context of lack of access to/patient refusal of blood products.

Facilitating better postnatal care with women-held documents in The Gambia: a mixed-methods study.

BACKGROUND: Women-held documents are a basic component of continuity of maternity care. The use and completion of women-held documents following discharge could improve treatment and care for postnatal women. Using a mixed-methods study design, we aimed to assess the number, type, quality and completeness of women-held discharge documents, identify factors contributing to document completeness and facilitators or barriers for effective use of the documents. METHODS: Documents given to women at discharge from three hospitals in the Greater Banjul Area, The Gambia, were reviewed for content and quality. All women completed a questionnaire on the use of the documents. Poisson regression was used to estimate factors predicting document completion. Semi-structured interviews (n = 21) and focus groups (n = 2) were carried out with healthcare professionals (HCPs). RESULTS: Nearly all (n = 211/212; 99%) women were given a document to take home. The most complete document (maternal record) had on average 17/26 (65%) items completed and 10% of women held an illegible document. None of the women's sociodemographic or clinical characteristics predicted document completeness. The following facilitators for effective use of documents were identified from the women's responses to the questionnaire and interviews with HCPs: 94% of women thought written information is important, 99% plan to have postnatal check-ups and 67% plan to use their documents, HCPs understand the importance of the documents and were familiar with the document's use and content. The following barriers for effective use of documents were identified: HCPs had too many women-held documents to complete at discharge, there is no national protocol and HCPs think women do not understand the documents due to a lack of education and that women often lose or forget their documents. CONCLUSIONS: Women-held documents are well established in The Gambia; though quality and completeness needs improving. Future research should determine the impact of using only one document at discharge, protocols and training on completeness, among other outcomes, and on ways to ensure all women are using the documents for their postnatal care.

The content and completeness of women-held maternity documents before admission for labour: A mixed methods study in Banjul, The Gambia.

BACKGROUND: Women-held maternity documents are well established for enabling continuity of maternity care worldwide, with the World Health Organisation (WHO) recommending their use in effective decision-making. We aimed to assess the presence, content and completeness of women-held maternity documents at admission to hospitals in The Gambia, and investigate barriers and facilitators to their completion. METHODS: We interviewed 250 women on maternity wards of all 3 Banjul hospitals and conducted content analysis of documentation brought by women on admission for their completeness against WHO referrals criteria. Logistic regression models were used to estimate the odds of the minimum criteria being met. Two focus groups and 21 semi-structured interviews (8 doctors, 8 midwives and 5 nurses) were conducted with healthcare practitioners to explore barriers and facilitators to documented clinical information availability on admission. FINDINGS: Of the women admitted, all but 10/250 (4%) brought either a maternity card or a structured referral sheet. Of all forms of documentation, women most frequently brought the government-issued maternity card (235/250, 94%); 16% of cards had all 9 minimum criteria completed. Of the 79 referred women, 60% carried standardised referral forms. Only 30% of 97 high-risk women had risk-status recorded. Women were less likely to have documents complete if they were illiterate, had not attended three maternity appointments, or lived more than one hour from hospital. During qualitative interviews, three themes were identified: women as agents for transporting information and documents (e.g. remembering to bring maternity cards); role of individual healthcare professionals' actions (e.g. legibility of handwriting); system and organisational culture (e.g. standardised referral guidelines). CONCLUSION: Women rarely forgot their maternity card, but documents brought at admission were frequently incomplete. This is a missed opportunity to enhance handover and quality of care, especially for high-risk women. National guidelines were recognised by providers as needed for good document keeping and would enhance the women-held maternity documents' contribution to improving both safety and continuity of care.

Evaluation of the Microvascular Blood Flow, Oxygenation, and Survival of Tarsoconjunctival Flaps Following the Modified Hughes Procedure.

PURPOSE: For the modified Hughes procedure, a tarsoconjunctival flap from the upper eyelid is used to reconstruct large, full-thickness, lower eyelid defects. The conjunctival pedicle is divided once vascularization is deemed to be adequate. The importance of maintaining a flap pedicle to ensure adequate perfusion of the graft has been questioned. The purpose of the study was to investigate the microvascular blood flow, oxygenation, and survival of a tarsoconjunctival flap in an experimental porcine model of the modified Hughes procedure. METHODS: The modified Hughes procedure was performed in 9 pigs. Microvascular blood flow was measured by laser Doppler velocimetry. Tissue oxygenation was measured using a Licox system, and tissue survival was determined by analyzing histologic sections of biopsy specimens from the lower edge of the flap. RESULTS: Blood flow and the oxygenation of the tissue decreased gradually during dissection and advancement of the tarsoconjunctival flap. At the time when the flap was sutured into place, there was virtually no blood flow or oxygenation of the tissue. However, flap survival did not seem to be compromised, as shown by the absence of pyknotic cell nuclei necrosis in the biopsy specimens, 12 hours after the procedure. CONCLUSIONS: The pedicle of the tarsoconjunctival flap does not seem to contribute to the nourishment of the tarsoconjunctival flap. Nourishment may be supplied by the rich vascularization of the remaining eyelid and tear film. If this is the case, single-stage grafting of a free tarsal plate may be performed, thus avoiding the eyelid-sharing stage of the procedure, without compromising the survival of the graft.

Use of bacteria- and fungus-binding mesh in negative pressure wound therapy provides significant granulation tissue without tissue ingrowth.

OBJECTIVE: Bacteria- and fungus-binding mesh traps and inactivates bacteria and fungus, which makes it interesting, alternative, and wound filler for negative pressure wound therapy (NPWT). The aim of this study was to compare pathogen-binding mesh, black foam, and gauze in NPWT with regard to granulation tissue formation and ingrowth of wound bed tissue in the wound filler. METHODS: Wounds on the backs of 8 pigs underwent 72 hours of NPWT using pathogen-binding mesh, foam, or gauze. Microdeformation of the wound bed and granulation tissue formation and the force required to remove the wound fillers was studied. RESULTS: Pathogen-binding mesh produced more granulation tissue, leukocyte infiltration, and tissue disorganization in the wound bed than gauze, but less than foam. All 3 wound fillers caused microdeformation of the wound bed surface. Little force was required to remove pathogen-binding mesh and gauze, while considerable force was needed to remove foam. This is the result of tissue growth into the foam, but not into pathogen-binding mesh or gauze, as shown by examination of biopsy sections from the wound bed. CONCLUSIONS: This study shows that using pathogen-binding mesh as a wound filler for NPWT leads to a significant amount of granulation tissue in the wound bed, more than that with gauze, but eliminates the problems of ingrowth of the wound bed into the wound filler. Pathogen-binding mesh is thus an interesting wound filler in NPWT.

Comparison of bacteria and fungus-binding mesh, foam and gauze as fillers in negative pressure wound therapy--pressure transduction, wound edge contraction, microvascular blood flow and fluid retention.

Bacteria- and fungus-binding mesh binds with and inactivates bacteria and fungus, which makes it an interesting alternative, wound filler for negative pressure wound therapy (NPWT). This study was conducted to compare the performance of pathogen-binding mesh, foam and gauze as wound fillers in NPWT with regard to pressure transduction, fluid retention, wound contraction and microvascular blood flow. Wounds on the backs of 16 pigs were filled with pathogen-binding mesh, foam or gauze and treated with NPWT. The immediate effects of 0, -40, -60, -80 and -120 mmHg, on pressure transduction and blood flow were examined in eight pigs using laser Doppler velocimetry. Wound contraction and fluid retention were studied during 72 hours of NPWT at -80 and -120 mmHg in the other eight pigs. Pathogen-binding mesh, gauze and foam provide similar pressure transduction to the wound bed during NPWT. Blood flow was found to decrease 0.5 cm laterally from the wound edge and increase 2.5 cm from the wound edge, but was unaltered 5.0 cm from the wound edge. The increase in blood flow was similar with all wound fillers. The decrease in blood flow was more pronounced with foam than with gauze and pathogen-binding mesh. Similarly, wound contraction was more pronounced with foam, than with gauze and pathogen-binding mesh. Wound fluid retention was the same in foam and pathogen-binding mesh, while more fluid was retained in the wound when using gauze. The blood flow 0.5-5 cm from the wound edge and the contraction of the wound during NPWT were similar when using pathogen-binding mesh and gauze. Wound fluid was efficiently removed when using pathogen-binding mesh, which may explain previous findings that granulation tissue formation is more rapid under pathogen-binding mesh than under gauze. This, in combination with its pathogen-binding properties, makes this mesh an interesting wound filler for use in NPWT.

The effects of variable, intermittent, and continuous negative pressure wound therapy, using foam or gauze, on wound contraction, granulation tissue formation, and ingrowth into the wound filler.

OBJECTIVE: Negative pressure wound therapy (NPWT) is commonly used in the continuous mode. Intermittent pressure therapy (IPT) results in faster wound healing, but it often causes pain. Variable pressure therapy (VPT) has therefore been introduced to provide a smooth transition between 2 different pressure environments, thereby maintaining the negative pressure environment throughout the therapy. The aim of the present study was to examine the effects of IPT and VPT on granulation tissue formation. METHOD: A peripheral wound in a porcine model was treated for 72 hours with continuous NPWT (-80 mm Hg), IPT (0 to -80 mm Hg), or VPT (-10 to -80 mm Hg), using foam or gauze as wound filler. Wound contraction and force to remove the wound filler were measured. Biopsies from the wound bed were examined histologically for granulation tissue formation. RESULTS: Intermittent pressure therapy and VPT produced similar results. Wound contraction was more pronounced following IPT and VPT than continuous NPWT. Intermittent pressure therapy and VPT resulted in the formation of more granulation tissue than continuous NPWT. Leukocyte infiltration and tissue disorganization were more prominent after IPT and VPT than after continuous NPWT. Granulation tissue grew into foam but not into gauze, regardless of the mode of negative pressure application, and less force was needed to remove gauze than foam. CONCLUSIONS: Wound contraction and granulation tissue formation is more pronounced following IPT and VPT than continuous NPWT. Granulation tissue grows into foam but not into gauze. The choice of negative pressure mode and wound filler is crucial in clinical practice to optimize healing while minimizing pain.

Negative pressure wound therapy-associated tissue trauma and pain: a controlled in vivo study comparing foam and gauze dressing removal by immunohistochemistry for substance P and calcitonin gene-related peptide in the wound edge.

Pain upon negative pressure wound therapy (NPWT) dressing removal has been reported and is believed to be associated with the observation that granulation tissue grows into foam. Wound tissue damage upon removal of the foam may cause the reported pain. Calcitonin gene-related peptide (CGRP) and substance P are neuropeptides that cause inflammation and signal pain and are known to be released when tissue trauma occurs. The aim of this controlled in vivo study was to compare the expression of CGRP and substance P in the wound bed in control wounds and following NPWT and foam or gauze dressing removal. Eight pigs with two wounds each were treated with open-pore structure polyurethane foam or AMD gauze and NPWT of 0 (control) or -80 mm Hg for 72 hours. Following removal of the wound filler, the expression of CGRP and substance P was measured, using arbitrary units, in sections of biopsies from the wound bed using immunofluorescence techniques. Substance P and CGRP were more abundant in the wound edge following the removal of foam than of gauze dressings and least abundant in control wounds. The immunofluorescence staining of the wound edge for CGRP was 52 ± 3 au after the removal of gauze and 97 ± 5 au after the removal of foam (P <0.001). For substance P, the staining was 55 ± 3 au after gauze removal and 95 ± 4 au after foam removal (P <0.001). CGRP and substance P staining was primarily located to nerves and leukocytes. The increase in CGRP and substance P immunofluorescence was especially prominent in the dermis but also was seen in subcutaneous and muscle tissue. Using gauze may be one way of reducing NPWT dressing change-related pain. New wound fillers designed to optimize granulation tissue formation and minimize pain issues presumably will be developed in the near future.

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia.

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1ß and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1ß immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.

Tumor necrosis factor and its receptors in the neuroretina and retinal vasculature after ischemia-reperfusion injury in the pig retina.

PURPOSE: Numerous studies have been performed aimed at limiting the extent of retinal injury after ischemia, but there is still no effective pharmacological treatment available. The aim of the present study was to examine the role of tumor necrosis factor (TNF)α and its receptors (TNF-R1 and TNF-R2), especially considering the neuroretina and the retinal vasculature since the retinal blood vessels are key organs in circulatory failure. METHODS: Retinal ischemia was induced in pigs by elevating the intraocular pressure to 80 mmHg in one eye, while the other eye served as a control (sham-operated). One hour of ischemia was followed by 5 or 12 h of reperfusion. Retinal circulation was examined in vivo by fundus imaging and fluorescein angiography. TNF-α levels were measured in the vitreous using an angiogenesis antibody array test. The presence and amounts of TNF-α, TNF-R1, and TNF-R2 were investigated in the neuroretina and in the retinal blood vessels, using immunofluorescence staining and real-time PCR techniques. RESULTS: Fundus imaging showed obstructed blood flow when ischemia was induced, and reperfusion was clearly visualized using fluorescein angiography. Ischemia resulted in elevated levels of TNF-α protein in the vitreous and TNF-α mRNA in the neuroretina. TNF-α immunofluorescence staining was localized to the Müller cells and the outer plexiform layer of the neuroretina. The expression of TNF-R1 and TNF-R2 mRNA was increased in both the neuroretina and retinal arteries following ischemia-reperfusion. Immunofluorescence double staining for TNF-R1 and either smooth muscle actin or 4',6-diamidino-2-phenylindole (DAPI) indicated expression in the cell membranes of the vascular smooth muscle cells. Double staining with TNF-R1 and calbindin showed localization to the horizontal cells in the outer plexiform layer of the neuroretina. CONCLUSIONS: Retinal ischemia results in increased expression of TNF-α and its receptors (TNF-R1 and TNF-R2). Cellular signaling pathways involving TNF may be important in the development of retinal injury following ischemia and thus an interesting target for future development of pharmacological therapeutics.

Micro- and macromechanical effects on the wound bed of negative pressure wound therapy using gauze and foam.

Negative pressure wound therapy (NPWT) results in 2 types of tissue deformation, macrodeformation (ie, wound contraction) and microdeformation (ie, the interaction of tissue and dressing on a microscopic level). These effects have been delineated for one type of wound filler, foam, but not for gauze. The mechanical deformation initiates a signaling cascade which ultimately leads to wound healing. The aim of the present study was to examine the effect of gauze and foam on macro- and microdeformation during treatment with negative pressure. An in vivo porcine peripheral wound model was used. NPWT was applied for 72 hours at 0, -75, and -125 mm Hg, using either foam or gauze as wound filler. The mechanical effects of NPWT were examined by measuring the wound surface area reduction and by histologic analysis of the wound bed tissue. Similar degrees of wound contraction (macrodeformation) were seen during NPWT regardless if foam or gauze was used. After negative pressure had been discontinued, the wound stayed contracted. There was no difference in wound contraction between -75 and -125 mm Hg. Biopsies of the wound bed revealed a repeating pattern of wound surface undulations and small tissue blebs ("tissue mushrooms") were pulled into the pores of the foam dressing and the spaces between the threads in the gauze dressing (microdeformation). This pattern was obvious in wounds treated both with foam and gauze, at atmospheric pressure (0 mm Hg) as well as at subatmospheric pressures (-75 and -125 mm Hg). The degrees of micro- and macrodeformation of the wound bed are similar after NPWT regardless if foam or gauze is used as wound filler.

Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion.

PURPOSE: The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. The results were compared to those of the sham- operated fellow eye. The retinal arteries and neuroretina were isolated separately and examined. Tissue morphology and DNA fragmentation were studied using histology. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, c-junNH(2)-terminal kinases (JNK), and c-jun protein and mRNA expression were examined using immunofluorescence staining, western blot, and real-time PCR techniques. RESULTS: Pyknotic cell nuclei, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, and glial fibrillary acidic protein mRNA expression were increased in ischemia, suggesting injury. Phosphorylated ERK1/2 protein levels were increased in the neuroretina following ischemia, while mRNA levels were unaltered. p38 protein and mRNA levels were not affected by ischemia. Immunofluorescence staining for phosphorylated p38 was especially intense in the retinal blood vessels, while only weak in the neuroretina. Phosphorylated JNK protein and mRNA were slightly decreased in ischemia. Phosphorylated c-jun protein and mRNA levels were higher in the neuroretina after ischemia-reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion alters expression of mitogen-activated protein kinases, particularly ERK1/2, in the neuroretina and retinal arteries. The development of pharmacological treatment targeting these intracellular transduction pathways may prevent injury to the eye following retinal circulatory failure.

Protein kinase C in porcine retinal arteries and neuroretina following retinal ischemia-reperfusion.

PURPOSE: Identification of the intracellular signal-transduction pathways activated in retinal ischemia may be important in revealing novel pharmacological targets. To date, most studies have focused on identifying neuroprotective agents. The retinal blood vessels are key organs in circulatory failure, and this study was therefore designed to examine the retinal vasculature separately from the neuroretina. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. Protein kinase C (PKC)alpha, PKCbeta1, and PKCbeta2 mRNA levels, and protein expression were determined using real-time PCR, western blot, and immunofluorescence staining techniques. RESULTS: The retinal arteries could easily be dissected free and studied separately from the neuroretina in this porcine model. The PKCalpha, PKCbeta1, and PKCbeta2 mRNA levels tended to be lower in ischemia-reperfused than in sham-operated eyes in both the retinal arteries and the neuroretina. This was most prominent after 5 h, and less pronounced after 12 h and 20 h of reperfusion. Likewise, the protein levels of PKCalpha, PKCbeta1, and PKCbeta2 were slightly lower following ischemia-reperfusion when compared to sham-operated eyes. PKCalpha, PKCbeta1, and PKCbeta2 immunostaining were observed in bipolar cells of the neuroretina and in endothelial cells, and to a low extent in the smooth muscle layer, of the retinal arteries. CONCLUSIONS: Retinal ischemia followed by reperfusion results in lower levels of PKC in both the neuroretina and retinal arteries. New targets for pharmacological treatment may be found by studying the retinal vasculature so as to identify the intracellular signal-transduction pathways involved in the development of injury following retinal circulatory failure.

Changes in proteasome structure and function caused by HAMLET in tumor cells.

BACKGROUND: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. METHODOLOGY/PRINCIPAL FINDINGS: HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. CONCLUSIONS/SIGNIFICANCE: The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

Can misfolded proteins be beneficial? The HAMLET case.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

Pressure transduction to the thoracic cavity during topical negative pressure therapy of a sternotomy wound.

The present study was performed to examine pressure transduction to the thoracic cavity during topical negative pressure (TNP) therapy of a sternotomy wound. Seven pigs underwent median sternotomy. Pressure transduction catheters were placed on the anterior surface of the heart (under the foam), in the pericardium (under the heart), in the left pleura and in the oesophagus at the level of the heart. The wound was sealed as for TNP therapy. The vacuum source was set to deliver negative pressures between -50 and -200 mmHg. The pressure on the anterior surface of the heart changed in a linear relationship with the applied negative pressure and was slightly lower than the applied negative pressure (-102 +/- 9 mmHg at delivered -125 mmHg). Further down in the thoracic cavity, in the pericardium (under the heart), in the left pleura and in the oesophagus, the wound pressure was only slightly affected by TNP therapy. In conclusion during TNP therapy, negative pressure is effectively transmitted to anterior portions of the heart. This may explain our recent findings that TNP increases microvascular blood flow in the myocardium. The pressure difference between the anterior and the posterior portions of the heart causes the right ventricle to be sucked up towards the posterior parts of the sternum, where it might be exposed to the sharp edges of the sternal bone, which may result in heart injury.