Publications by authors named "Lotta Gustafsson"

33 Publications

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

PLoS One 2020 6;15(3):e0230063. Epub 2020 Mar 6.

Institute of Applied Health Research: University of Birmingham, Birmingham Clinical Trials Unit College of Medical and Dental Sciences, Birmingham, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230063PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059937PMC
June 2020

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

Ophthalmic Plast Reconstr Surg 2016 Nov/Dec;32(6):468-472

*Department of Ophthalmology, Clinical Sciences, Lund, Lund University and Skåne University Hospital, Lund, Sweden and †Department of Dermatology, Clinical Sciences, Lund, Lund University and Skåne University Hospital, Lund, Sweden.

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.
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http://dx.doi.org/10.1097/IOP.0000000000000598DOI Listing
February 2017

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

Eplasty 2014 17;14:e3. Epub 2014 Jan 17.

Departments of Ophthalmology, Lund University and Skåne University Hospital, Lund, Sweden.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899807PMC
February 2014

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.

Int Wound J 2013 Oct 21;10(5):597-605. Epub 2012 Jun 21.

Department of Ophthalmology, Lund University, Lund, Sweden Department of Cardiothoracic Surgery, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1111/j.1742-481X.2012.01029.xDOI Listing
October 2013

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.

Eplasty 2012 24;12:e5. Epub 2012 Jan 24.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266212PMC
August 2012

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.

Ostomy Wound Manage 2011 Dec;57(12):30-5

Department of Ophthalmology, Lund University and Skåne University Hospital, Lund, Sweden.

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.
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December 2011

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

Mol Vis 2010 Nov 6;16:2317-27. Epub 2010 Nov 6.

Department of Ophthalmology, Lund University, Sweden.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994763PMC
November 2010

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

J Ocul Biol Dis Infor 2010 May 27;3(1):20-9. Epub 2010 May 27.

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 1β 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-1β 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.
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http://dx.doi.org/10.1007/s12177-010-9050-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956450PMC
May 2010

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

Ann Plast Surg 2010 Jun;64(6):789-93

Department of Ophthalmology, Lund University Hospital, Lund, Sweden.

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.
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http://dx.doi.org/10.1097/SAP.0b013e3181ba578aDOI Listing
June 2010

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

Mol Vis 2010 Mar 10;16:392-407. Epub 2010 Mar 10.

Department of Ophthalmology, Lund University, Lund, Sweden.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838742PMC
March 2010

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

Mol Vis 2009 13;15:737-46. Epub 2009 Apr 13.

Department of Ophthalmology, Lund University, Sweden.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668768PMC
May 2009

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

PLoS One 2009 14;4(4):e5229. Epub 2009 Apr 14.

Department of Microbiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664966PMC
October 2009

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

Int J Cancer 2009 Mar;124(5):1008-19

Institute of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1002/ijc.24076DOI Listing
March 2009

Can misfolded proteins be beneficial? The HAMLET case.

Ann Med 2009 ;41(3):162-76

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, Lund, Sweden.

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.
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http://dx.doi.org/10.1080/07853890802502614DOI Listing
July 2009

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

Int Wound J 2008 Oct 19;5(4):579-84. Epub 2008 Sep 19.

Department of Medicine, Lund University Hospital, Lund, Sweden.

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.
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http://dx.doi.org/10.1111/j.1742-481X.2007.00425.xDOI Listing
October 2008

Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways.

BMC Cardiovasc Disord 2008 Sep 8;8:21. Epub 2008 Sep 8.

Department of Medicine, Lund University Hospital, Sweden.

Background: Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.

Methods: Human internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists.

Results: The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 microM bisindolylmaleimide I and 10 microM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 microM SB203580, 10 microM PD98059 and 10 microM SP600125, respectively).

Conclusion: In conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.
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http://dx.doi.org/10.1186/1471-2261-8-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553399PMC
September 2008

Topical negative pressure therapy of a sternotomy wound increases sternal fluid content but does not affect internal thoracic artery blood flow: assessment using magnetic resonance imaging.

J Thorac Cardiovasc Surg 2008 May;135(5):1007-13

Department of Medicine, Lund University Hospital, Lund, Sweden.

Objective: Topical negative pressure therapy has excellent healing effects in poststernotomy mediastinitis. Topical negative pressure therapy reduces bacterial counts, increases wound edge microvascular blood flow and granulation tissue formation, and facilitates healing. No study has yet been performed to examine the effect of topical negative pressure on the blood and fluid content in the sternal bone marrow, which is a crucial component in osteitis.

Methods: Eight pigs underwent median sternotomy, left internal thoracic artery harvesting, followed by topical negative pressure treatment. Magnetic resonance imaging was used to quantify both tissue fluid and/or blood content (T2-weighted short tau inversion recovery [T2-STIR]) and internal thoracic artery blood flow (flow quantification).

Results: Before application of topical negative pressure, the T2-STIR signal intensity ratio was lower for the left than for the right hemisternum (left, 1.3; right, 2.6), indicating lower levels of tissue fluid content on the left, devascularized side. On application of topical negative pressure, the T2-STIR signal intensity ratio increased immediately for both the sternal bone and the pectoral muscle (left hemisternum after 4 minutes of topical negative pressure: 2.3), leveled off after 4 minutes, and remained unchanged for the ensuing 40 minutes, suggesting movement of fluid and/or blood into the tissue of the wound edge. Topical negative pressure did not affect blood flow in the right internal thoracic artery.

Conclusions: T2-STIR measurements show that topical negative pressure increases sternotomy wound edge tissue fluid and/or blood content. Topical negative pressure creates a pressure gradient that presumably draws fluid from the surrounding tissue to the sternal wound edge and into the vacuum source. This "endogenous drainage" may be one possible mechanism by which osteitis is resolved by topical negative pressure in poststernotomy mediastinitis.
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http://dx.doi.org/10.1016/j.jtcvs.2007.09.070DOI Listing
May 2008

Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

Adv Exp Med Biol 2008 ;606:217-40

Department for Experimental Medical Sciences, Section for Lungbiology, Lund, Sweden.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.
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http://dx.doi.org/10.1007/978-0-387-74087-4_8DOI Listing
January 2008

Histone deacetylase inhibitors promote the tumoricidal effect of HAMLET.

Cancer Res 2007 Dec;67(23):11327-34

Institute of Laboratory Medicine, Section of Microbiology, Immunology, and Glycobiology, Lund University, Lund, Sweden.

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-1153DOI Listing
December 2007

PKC and MAPK signalling pathways regulate vascular endothelin receptor expression.

Eur J Pharmacol 2008 Feb 1;580(1-2):190-200. Epub 2007 Nov 1.

Department of Medicine, Lund University Hospital, Lund, Sweden.

Up-regulation of vascular endothelin type A (ET(A)) and type B (ET(B)) receptors are implicated in the pathogenesis of cardiovascular disease. Culture of arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for in detail delineation of the regulation of endothelin receptors. We hypothesize that protein kinase C (PKC) and mitogen-activated kinases (MAPK) are involved in the regulation of endothelin receptors. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology, real-time PCR and immunofluorescence techniques. Sarafotoxin 6c and endothelin ET-1 were used to examine the endothelin ET(A) and ET(B) receptor effects. The involvement of PKC and MAPK in the receptor regulation was examined by culture in the presence of antagonists. Organ culture resulted in increased sarafotoxin 6c and endothelin-1 contractions, endothelin ET(A) and ET(B) receptor immunofluorescence staining intensities and endothelin ET(B), but not ET(A), receptor mRNA levels. The general PKC inhibitors, bisindolylmaleimide I (10 microM) or Ro-32-0432 (10 microM), inhibited these effects. Also, the increase in sarafotoxin 6c contraction, endothelin ET(B) receptor and mRNA levels and endothelin ET(A) and ET(B) immunofluorescence staining intensities were inhibited by MAPK inhibitors for extracellular signal related kinases 1 and 2 (ERK1/2), PD98059 (10 microM), C-jun terminal kinase (JNK), SP600125 (10 microM), but not by p38 MAPK, SB203580 (10 microM). In conclusion, PKC and MAPK seem to be involved in the regulation of endothelin receptor expression in porcine coronary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin receptor changes in cardiovascular disease.
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http://dx.doi.org/10.1016/j.ejphar.2007.10.071DOI Listing
February 2008

Endothelin receptor-mediated vasodilatation: effects of organ culture.

Eur J Pharmacol 2008 Jan 3;579(1-3):233-40. Epub 2007 Oct 3.

Department of Medicine, Lund University Hospital, Lund, Sweden.

Culture of intact arteries is a frequently employed experimental model for investigating the mechanisms governing the regulation of vascular endothelin receptors. Endothelin type A (ET(A)) and type B (ET(B)) receptors on vascular smooth muscle cells are up-regulated in organ culture and the enhanced vasoconstriction mimics the changes that occur in cardiovascular disease. The effect of organ culture on endothelial dilatory endothelin ET(B) receptors is not known. We hypothesize that organ culture decreases the endothelin receptor-mediated dilatation and that this is one possible mechanism by which the effects of the endothelin in blood vessels are altered during culture. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology and immunofluorescence. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ET(A) and ET(B) receptor effects, and the antagonists, Nomega-nitro-l-arginine (l-NOARG) for nitric oxide (NO), indomethacin for prostaglandins and charybdotoxin in combination with apamin for endothelium-derived hyperpolarizing factor (EDHF), were used to study the endothelium-derived dilatory mediators. Organ culture induced up-regulation of the sarafotoxin 6c (ET(B) receptor agonist) and endothelin-1 (ET(A) receptor agonist) elicited vasoconstriction. The sarafotoxin 6c contraction was stronger after endothelium denudation, suggesting endothelium-dependent dilatation. The endothelin-1 contraction was not affected by endothelium denudation. The increase in sarafotoxin 6c contraction after removal of the endothelium was more pronounced before than after organ culture, suggesting down-regulated endothelial endothelin ET(B) receptors. Also, the immunofluorescence staining intensities for endothelial endothelin ET(B) receptors were higher before than after organ culture. Pre-incubation with inhibitors for dilatory mediators suggested that both NO and EDHF play a vasodilatory role, while prostaglandins are not involved. In conclusion, endothelial endothelin ET(B) receptors induce NO and EDHF mediated vasodilatation in porcine coronary arteries. In organ culture, endothelial endothelin ET(B) receptors are down-regulated, mimicking the changes that occur in cardiovascular disease. Down-regulation of endothelial endothelin ET(B) receptors may in part explain the increased endothelin ET(B) receptor-mediated vasoconstriction frequently studied in organ culture.
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http://dx.doi.org/10.1016/j.ejphar.2007.09.031DOI Listing
January 2008

Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

Int J Cancer 2007 Sep;121(6):1352-9

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

We studied if bladder cancers respond to HAMLET (human alpha-lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native alpha-lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.
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http://dx.doi.org/10.1002/ijc.22810DOI Listing
September 2007

Hemodynamic effects of vacuum-assisted closure therapy in cardiac surgery: assessment using magnetic resonance imaging.

J Thorac Cardiovasc Surg 2007 May;133(5):1154-62

Department of Medicine, Lund University Hospital, Lund, Sweden.

Objective: The hemodynamic effects of vacuum-assisted closure therapy in cardiac surgery are debated. The aim of the present study was to quantify cardiac output and left ventricular chamber volumes after vacuum-assisted closure using magnetic resonance imaging, which is known to be the most accurate method for quantifying these measures.

Methods: Six pigs had median sternotomy followed by vacuum-assisted closure treatment in the presence and absence of a paraffin gauze interface dressing. Cardiac output and stroke volume were examined using magnetic resonance imaging flow quantification (breath-hold and real-time). Chamber volumes were assessed using cine magnetic resonance imaging.

Results: Cardiac output and stroke volume decreased immediately after application of negative pressures of 75, 125, and 175 mm Hg (13% +/- 1% decrease in cardiac output). Interposition of 4 layers of paraffin gauze dressing over the heart during vacuum-assisted closure therapy resulted in a smaller decrease in cardiac output (8% +/- 1%).

Conclusions: Vacuum-assisted closure therapy results in an immediate decrease in cardiac output, although to a lesser extent than shown previously. Covering the heart with a wound interface dressing lessens the hemodynamic effects of vacuum-assisted closure.
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http://dx.doi.org/10.1016/j.jtcvs.2007.01.011DOI Listing
May 2007

Ceramide as a TLR4 agonist; a putative signalling intermediate between sphingolipid receptors for microbial ligands and TLR4.

Cell Microbiol 2007 May 11;9(5):1239-51. Epub 2007 Jan 11.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-22362 Lund, Sweden.

Mucosal Toll-like receptors (TLRs) respond to pathogens, but remain inert to the indigenous flora, suggesting that the TLRs can receive pathogen-specific signals. For example, TLR4 signalling is activated in CD14-negative epithelial cells by P-fimbriated, uropathogenic Escherichia coli, but not by lipopolysaccharide. The fimbriae use glycosphingolipids as recognition receptors and there is release of ceramide, which is the membrane-anchoring domain of the receptors. In this study, ceramide was identified as a TLR4 agonist and as a putative signalling intermediate between the glycosphingolipid recognition receptors and TLR4. Exogenous ceramide activated a TLR4-dependent epithelial cell response, as shown by exposing stably transfected TLR4-positive or -negative human embryonal kidney cells to C2 and C6 ceramide. A similar, TLR4-dependent response occurred after deliberate release of endogenous long-chained ceramide with sphingomyelinase. Microbial ligands with glycosphingolipid specificity (P fimbriae or the B subunit of Shiga toxin) were shown to increase the levels of ceramide and to trigger a TLR4-dependent response in epithelial cells. The results show that ceramide activates TLR4 signalling and suggest that this mechanism might allow pathogens to elicit mucosal TLR4 responses by perturbing sphingolipid receptors for virulence ligands like P fimbriae.
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http://dx.doi.org/10.1111/j.1462-5822.2006.00867.xDOI Listing
May 2007

Sternal stability at different negative pressures during vacuum-assisted closure therapy.

Ann Thorac Surg 2006 Sep;82(3):1063-7

Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Sweden.

Background: Vacuum-assisted closure (VAC) is a widely used therapy in patients with poststernotomy mediastinitis. The aim of this study was to evaluate sternal stability during VAC application at seven negative pressures (-50 to -200 mm Hg) in a porcine wound model.

Methods: Six pigs underwent median sternotomy and 2 steel wires were fixed at each sternal side and connected to a traction device. The device was connected to a force transducer linked to a force recorder. VAC therapy was applied to the wound. At each negative pressure, the length and width of the wound were measured before and after traction was started. Traction was increased stepwise up to 400 N.

Results: The diastasis induced by a certain lateral force was similar in wounds treated with -75, -125, and -175 mm Hg. At -75 mm Hg, a significant improvement (p < 0.01) in sternal stability was seen compared with the open-chest setting. This was not further improved at -125 or -175 mm Hg. High negative pressures (-150 to -200 mm Hg) in combination with a high lateral force (>200 N) increased the risk of separation of the foam from the wound edges, with air leakage or organ rupture as a result.

Conclusions: Our results suggest that low negative pressures (-50 to -100 mm Hg) stabilize the sternum as efficiently as high negative pressures (-150 to -200 mm Hg). Low negative pressures (-50 to -100 mm Hg) were more beneficial, however, because no air leakage or organ rupture was observed at these pressures.
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http://dx.doi.org/10.1016/j.athoracsur.2006.04.085DOI Listing
September 2006

Effect of vacuum-assisted closure on blood flow in the peristernal thoracic wall after internal mammary artery harvesting.

Eur J Cardiothorac Surg 2006 Jul 26;30(1):85-9. Epub 2006 May 26.

Department of Medicine, Lund University Hospital, Lund, Sweden.

Objective: Vacuum-assisted closure (VAC) is a recently introduced method for the treatment of poststernotomy mediastinitis. The aim was to examine the effects of VAC negative pressure on peristernal soft tissue blood flow after internal mammary artery harvesting.

Methods: Microvascular blood flow was measured using laser Doppler velocimetry in a porcine sternotomy wound model. The effect of VAC negative pressure on blood flow to the wound edge was investigated on the right side, where the internal mammary artery was intact, and on the left side, where the internal mammary artery had been removed.

Results: Before removal of the left internal mammary artery, the blood flow was similar in the right and left peristernal wound edges, 2.5 cm from the edge (27+/-4 perfusion units (PU) on the right side and 32+/-3 PU on the left side, in muscle tissue). When the left internal mammary artery was surgically removed, the blood flow on the left side decreased (19+/-3 PU, in muscle tissue), while the skin blood flow was not affected. VAC negative pressure induced an immediate increase in wound edge blood flow both on the right side (43+/-9 PU, in muscle tissue at -75 mmHg), where the internal mammary artery was intact, and on the left side, where the internal mammary artery had been removed (49+/-11 PU, in muscle tissue at -75 mmHg). The increase in blood flow was similar on both sides at -75 mmHg and at -125 mmHg.

Conclusions: The peristernal wound edge microvascular blood flow is decreased when the left internal mammary artery is removed. VAC therapy stimulates blood flow in the peristernal thoracic wall after internal mammary artery harvesting.
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http://dx.doi.org/10.1016/j.ejcts.2006.04.009DOI Listing
July 2006

HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy.

J Nutr 2005 May;135(5):1299-303

Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, Lund University, Sweden.

New cancer treatments should aim to destroy tumor cells without disturbing normal tissue. HAMLET (human alpha-lactalbumin made lethal to tumor cells) offers a new molecular approach to solving this problem, because it induces apoptosis in tumor cells but leaves normal differentiated cells unaffected. After partial unfolding and binding to oleic acid, alpha-lactalbumin forms the HAMLET complex, which enters tumor cells and freezes their metabolic machinery. The cells proceed to fragment their DNA, and they disintegrate with apoptosis-like characteristics. HAMLET kills a wide range of malignant cells in vitro and maintains this activity in vivo in patients with skin papillomas. In addition, HAMLET has striking effects on human glioblastomas in a rat xenograft model. After convection-enhanced delivery, HAMLET diffuses throughout the brain, selectively killing tumor cells and controlling tumor progression without apparent tissue toxicity. HAMLET thus shows great promise as a new therapeutic with the advantage of selectivity for tumor cells and lack of toxicity.
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http://dx.doi.org/10.1093/jn/135.5.1299DOI Listing
May 2005

Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid.

N Engl J Med 2004 Jun;350(26):2663-72

Institute of Laboratory Medicine, Department of Microbiology, Immunology, and Glycobiology, University of Lund, Lund, Sweden.

Background: We studied the effect on skin papillomas of topical application of a complex of alpha-lactalbumin and oleic acid (often referred to as human alpha-lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that alpha-lactalbumin-oleic acid kills transformed cells but not healthy, differentiated cells.

Methods: Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which alpha-lactalbumin-oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of alpha-lactalbumin-oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term follow-up data were obtained.

Results: In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the alpha-lactalbumin-oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with alpha-lactalbumin-oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with alpha-lactalbumin-oleic acid, and the time to resolution was shorter in the group originally assigned to receive alpha-lactalbumin-oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients.

Conclusions: Treatment with topical alpha-lactalbumin-oleic acid has a beneficial and lasting effect on skin papillomas.
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http://dx.doi.org/10.1056/NEJMoa032454DOI Listing
June 2004

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

Cancer Res 2004 Mar;64(6):2105-12

Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, University of Lund, Sölvegatan 23, 223-62 Lund, Sweden.

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.
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http://dx.doi.org/10.1158/0008-5472.can-03-2661DOI Listing
March 2004

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

Protein Sci 2003 Dec;12(12):2794-804

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366987PMC
http://dx.doi.org/10.1110/ps.0231003DOI Listing
December 2003