PubMed Health⌕ Search

Biomedical subjects

L Steinstraesser

Publications and source records attributed to L Steinstraesser.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of biodegradation of copolymer dermis substitutes in the dorsal skinfold chamber of balb/c mice.

PEGT/PBT-block-copolymer dermis substitutes were inserted into dorsal skinfold chambers of balb/c mice (n=36). Scaffolding matrices with 3 different pore diameters (pore diameter: <75 micro m, 75-212 micro m and 250-300 micro m) were analyzed on days 7, 14, and 21 post implantation by scanning electron and light microscopy. The quantification of matrix fragmentation was performed using image-analytical software analySIS(R). The fragmentation rate in scaffolding matrices with a pore size of < 75 micro m was observed to be higher than in matrices of larger pore sizes. Image-analytical evaluation over 21 days revealed a reduction of the copolymer matrix by approximately 32% for the <75 micro m matrices, 23% for the 75-212 micro m matrices and 18% for the matrices, where pore size ranged between 250 micro m and 300 micro m. Twenty-one days after implantation, the matrix pores of 75-212 micro m and 250-300 micro m scaffolds were totally filled by vascularized fibrous tissue. Contrarily, an increased formation of foreign-body giant cells was observed in matrices with pore size <75 micro m. The pore size of the scaffolding PEGT/PBT dermis substitutes affects their degradative behaviour in vivo.

Animals↗
PubMed2006

Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin.

BACKGROUND: The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds. METHODS: Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for beta-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine 2000, (2) FuGENE 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation. RESULTS: Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein). CONCLUSIONS: It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene.

Adenoviridae↗
PubMed2006

[Differential diagnosis of "sterile" phlegmonous hand infections].

INTRODUCTION: Bacterial infections represent a large proportion of emergencies in hand surgery. In some cases, pyoderma gangrenosum and mycobacterial infection may present with the same symptoms of swelling, pain, and purulent secretion. In these cases, operative treatment would be harmful. Therefore two cases-pyoderma gangrenosum and tuberculosis-are presented here in relation to common bacterial hand infection. METHODS: Using two case reports of diseases that only rarely affect the hands, their relevance to differential diagnosis is shown with reference to the literature. RESULTS: In both cases, we found clinical symptoms of bacterial hand infection with negative bacterial smear tests. After several debridements, pyoderma gangrenosum of the dorsum of the hand was diagnosed in one patient after pyodermiform lesions at the thigh and the nasal septum were detected and pre-existing colitis ulcerosa was taken into consideration. Corticoid therapy induced complete remission. The second patient with similar clinical symptoms had been operated on at another hospital several times before being transferred to our institution. The presumptive diagnosis of pyoderma gangrenosum was made, and under treatment with prednisone the symptoms quickly improved. After 2 weeks, the wound conditions and the patient's condition rapidly worsened. Following amputation at the upper arm level, the patient died of septic multiple organ failure. Autopsy studies revealed tuberculous sepsis originating from the hand. DISCUSSION: Patient history should be evaluated carefully because of its value to correct diagnosis. In case of negative smear tests, especially from immunocompromised, elderly patients and in patients with a history of pulmonary tuberculosis, Ziehl-Neelsen staining should be obtained. In case of multilocular affection or pre-existing chronic inflammatory bowel disease, the presumptive diagnosis of pyoderma gangrenosum can be confirmed by biopsies from the lesions margin. In both cases, unnecessary traumatizing operations could thus be avoided and treatment optimized.

Adult↗
PubMed2005

Activity of histone H1.2 in infected burn wounds.

OBJECTIVES: Infections with multidrug-resistant microorganisms (e.g. Pseudomonas aeruginosa and Staphylococcus aureus) cause immense complications in wound care and in the treatment of immunosuppressed patients. Like most antimicrobial peptides, histones are relatively small polycationic proteins located in each eukaryotic nucleus, which naturally supercoil DNA. The aim of this study was to investigate the in vitro and in vivo activity of histone H1.2 in infected burn wounds and its potential toxicity. METHODS: To characterize the antimicrobial properties of histone H1.2 against potential causative organisms of burn wound infections, the in vitro radial diffusion assay and modified NCCLS microbroth dilution MIC assay were carried out. Haemolytic and cytotoxic properties were determined in human red blood cells and primary human keratinocytes. In vivo antimicrobial activity was tested in an infected rat burn model with P. aeruginosa (ATCC 27853). All results were compared with the naturally occurring broad-spectrum antimicrobial peptide protegrin-1 and with antibiotics clinically used against the corresponding bacteria. RESULTS: Human histone H1.2 exerted good antimicrobial activity against all tested microorganisms without significant haemolytic activity. Surprisingly, histone H1.2 showed cytotoxicity with an LD50 of 7.91 mg/L in primary human keratinocytes. The in vivo burn model data revealed a significant three-fold higher reduction in bacterial counts within 4 h compared with carrier control. CONCLUSIONS: These findings indicate that histone H1.2 is a potential candidate for use as a local and, because of its low haemolytic activity, systemic antimicrobial agent. However, further investigations are needed to specify the cytotoxicity and the dose-response relationship for histone H1.2.

Animals↗
PubMed2005

A qualitative and quantitative analysis of protein loss in human burn wounds.

INTRODUCTION: It is well known that in patients suffering from major burn injuries of more than 15% of total body surface area (TBSA), capillary leak and loss of proteins including immunoglobulins (Ig) lead to cardiovascular failure and significantly elevated risk of infections. However, knowledge of the resulting protein profiles is limited. In order to elucidate quantitative and qualitative protein loss in human burn wounds we compared wound fluid (WF) protein content with serum protein levels. MATERIAL AND METHODS: Eleven patients suffering from second degree burns of 18-68% TBSA were enrolled in the study. Immediately after admission burn wounds were enclosed in cutaneous vinyl wound chambers covering a 2.25 cm(2) wound surface area. WF and serum samples were harvested every 8 h with a follow up of 48 h and analyzed for total protein content, albumin and the immunoglobulins A, E, G and M. RESULTS: Protein levels in serum were significantly lower as compared to physiological levels while WF protein levels were elevated and remained high. Total protein (TP) and albumin (AL) accumulated in high concentrations on the wound surface (average accumulation on 10% burnt TBSA within 8 h: TP=16.59+/-8.86 g; AL=12.39+/-5.87 g). The albumin fraction in WF showed increasing values (24 h: 69%; 32 h: 86%) although the serum albumin fraction remained nearly unchanged (55%). Peak values were initially found for all immunoglobulins both in serum and WF. IgA, E and M reached a steady state 32 h post-trauma, whereas IgG continuously decreased until 40 h. IgG values in serum were significantly below physiological levels at all time points. CONCLUSIONS: This study qualifies and quantifies a significant protein loss in second degree burn wounds. Protein concentrations in wound fluid correlate highly with serum concentrations until 48 h post-burn. A patient's entire amount of serum proteins accumulates in wound fluid in a 20% TBSA burn within approximately 24h. In contrast to capillary leak theory proteins and immunoglobulins extravasate to wound fluid even after 48 h post-trauma.

Adult↗
PubMed2005

New model for in vivo investigation after microvascular breakdown in burns: use of intravital fluorescent microscopy.

BACKGROUND: The breakdown of skin microcirculation is assumed to play a key role in the pathophysiology after burn injury. The aim of the present study was to develop a burn model, which allows repetitive quantitative in vivo analysis of the microcirculation after a burn injury, focusing on the interaction between leukocytes and the endothelium. MATERIALS AND METHODS: Experiments were carried out on male hairless mice. Deep partial thickness burns were inflicted with a no-touch-technique to the ears. Intravital fluorescent microscopy in combination with FITC-dextran as a plasma marker was used to assess microcirculatory parameters. Leukocytes were stained with rhodamine 6G. Preburn baseline data was obtained before as well as 1, 3, 7 and 14 days subsequent to the burn injury. RESULTS: The non-perfused area decreased significantly over the observed period and perfusion was almost completely restored at day 14. The functional vessel density was characterized by reduction of perfused vessels immediately after burn and an increase after 24h. Leukocyte endothelium interaction significantly increased immediately after injury; baseline values were reached 1 day later. The extravasation of the plasma marker into the surrounding tissue increased immediately after burn, decreased at day 1 and remained at this level during the following observation time. The venular as well as the arterial blood flow increased immediately subsequent to the burn injury, decreased after 1 day and reached baseline values at day 3. CONCLUSION: The presented burn model allows quantitative assessment of the dynamics of microcirculatory disturbances after thermal trauma by high quality visualization of both plasma stained microvessels and leukocyte-endothelium interaction.

Animals↗
PubMed2005

Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections.

Host defense peptides (HDP) are naturally occurring effector molecules of the innate immune system, which might be an alternative to currently used antibiotics. The objective of this study was to investigate the efficiency of transient cutaneous adenoviral transfection with human cathelicidin hCAP-18/LL-37 in infected burn wounds. Specific transgene expression was analyzed in vitro on mRNA and protein level using real-time PCR and Western-blot. Male Sprague-Dawley rats (n=40) received a second degree scald burn on both flanks (5% BSA), which were inoculated with 10(8) colony-forming units (CFU) Pseudomonas aeruginosa. Two days later, rats were randomized into the following groups: (1) adenoviral delivery of LL-37 (Ad5-hCAP-18, n=10), (2) synthetic host defense peptide LL-37 (1 mg; n=10), (3) carrier control (PBS, n=10) and (4) empty-virus control (Ad5-LacZ, n=10). Agents were injected intradermally and subcutaneously into both flanks. After either 2 or 7 days, skin samples were harvested and homogenized. CFU per gram tissue were determined. The hCAP-18/LL-37 expression was confirmed by real-time PCR and localized using in situ hybridization. In vitro transfection of cutaneous cells delivered a specific response on mRNA production. Western blot analysis revealed protein expression of hCAP-18/LL-37 in conditioned medium and cell pellet. The host defense peptide LL-37 was detectable after cleavage of the inactive pro-form hCAP-18/LL-37 with human elastase. Ad5-hCAP-18 showed a significant bacterial inhibition of approximately 10 000 fold compared to the control group (P<0.001) and 1000-fold (P<0.001) compared to the synthetic HDP LL-37 7 post-transfection. No inhibition was observed for the carrier or empty-virus control. Real-time PCR and in situ hybridization confirmed expression of hCAP-18/LL-37. In conclusion, transient cutaneous adenoviral delivery of the host defense peptide hCAP-18/LL-37 is significantly more effective than administration of synthetic host defense peptides and might be a potential adjunct for wound treatment in the near future.

Adenoviridae↗
PubMed2005

[Serious complications of injections--retrospective analysis of incidences, complication-management, prophylaxis and economic aspects].

The parenteral drug application is a routinely used method in all medical disciplines. Intramuscular, intraarticular, intravenous injections and infusions can cause local complications such as abscesses, articular infections or paravasates. These local complications can lead to bacteraemia, sepsis and may lead to multiple organ failure associated with high morbidity and mortality. Although these complications are rare, they are sometimes disastrous and result in life threatening clinical conditions. During a retrospective analysis (review period 1998-2002) 24 patients were admitted and hospitalized in our department. Within this report we demonstrate 7 patients with fatal complications after injections. In the majority of cases minor patients' complaints were proceeding before major complications were present. A long and expensive treatment period with multiple surgical interventions ends up in functional disabilities and unsatisfactory aesthetic results. Instead of delayed surgical treatment immediate radical surgical care is crucial to prevent disastrous complications. In case of the inability of sufficient debridement, amputations are sometimes indicated in the sense of "life before limb". Besides the consequences for the patient these disastrous complications have a high socioeconomic impact and result in reduced reimbursement for the hospital stay.

Abscess↗
PubMed2004

The impact of topical antiseptics on skin microcirculation.

AIM: Antiseptics are commonly used in clinical practice to disinfect tissue and to avoid infections. However, topical antiseptics are assumed to have an influence on skin microcirculation, per se. Thus, the aim of the study was to analyse the influence of topically applied antiseptics on the microcirculation of intact skin in vivo. MATERIALS AND METHODS: The investigation was carried out on ears of male hairless mice (SKH-1hr, n = 25). The influence of four antiseptics was examined. Sodium chloride 0.9% served as control. An alcohol-based solution with a mixture of ethanol, 2-propanol and purified water (Softasept), an antiseptic with octenidine dihydrochloride and phenoxyethanol as the main active agents (Octenisept), as well as hexamethylenbiguanide (Lavasept) and 70% ethanol were tested. Intravital fluorescence microscopy in combination with intravenous injection of the fluorescence dyes FITC-Dextran as plasma marker and Rhodamine 6G (leukocyte staining) allowed a quantitative analysis of standard microcirculatory parameters (vessel diameter, functional capillary density, red blood cell velocity, FITC-leakage and leukocyte endothelium interaction). Recordings of the microcirculation in several regions of interest (ROI) were made prior to application and after 10 min exposure time and 60 min after the baseline data. Data were evaluated off-line with aid of computer assisted analysis. RESULTS: The diameter of arterioles decreased after the treatment with the alcoholic solutions. The other two antiseptics (Octenisept and Lavasept) caused a significant increase. Functional capillary density (FCD) was significantly reduced after application of ethanol and Softasept. There was no reduction of FCD following application of Octenisept. After treatment with ethanol and Softasept there was a significant decrease in red blood cell velocity (RBCV). The use of Lavasept revealed a decrease of FCD and RBCV. In the Octenisept treated group RBCV shows a mild increase after 10 minutes. The application of ethanol, Softasept and Lavasept was characterized by a significant increase of leukocyte endothelium interaction (LEI). After treatment with saline and Octenisept LEI remained constant. All used antiseptics except of Octenisept caused a significant leakage of FITC-Dextran. CONCLUSION: The antiseptics used in this study all showed an influence on skin microcirculation. As expected, our findings show that the alcoholic solutions are most aggressive to skin microcirculation.

Administration, Topical↗
PubMed2004

Host defense peptides in burns.

Overuse of antibiotics and failure to apply basic infection control policies and procedures have contributed to the increasing multi-drug resistance of many nosocomial pathogens. The alarming increase of multi-drug-resistant bacteria (e.g. Pseudomonas aeruginosa, methicilin-resistant Staphylococci, vancomycin-resistant Enterococci) causes infected wounds associated with high mortality and morbidity in burned patients and focuses attention on the need for better treatment and prevention of wound infections. The review points out and discusses some emerging alternatives to antibiotics used in clinical practice, with special emphasis on the role of the innate immune response and potential application of human host defense peptides in thermal injury.

Antimicrobial Cationic Peptides↗
PubMed2004

[Gorham-Stout disease: report of a case affecting the right hand with a follow-up of 24 years].

Gorham-Stout disease is a rare idiopathic syndrome with distinctive clinical, pathologic and radiologic features. It is a variant form of osseus angiomatosis associated with massive osteolysis of bone. Usually appearing after trauma, the disease is described to occur at any age. Especially in case of thoracic involvement (chylothorax), lethal outcomes are reported. In Medline, about 200 cases have been described. A patient with osteolysis of the right hand following contusion at the age of two years is reported. Despite radiotherapy and repeated bone grafting, the osteolysis progressed until today (24 years). The pathologic features and various treatment methods for hand involvement are discussed.

Adult↗
PubMed2003

[No problem with liposuction?].

Subcutaneous liposuction in tumescent technique is the most frequent aesthetic plastic procedure in the United States. In Germany, nearly 250,000 liposuctions are done per year by a variety of surgical and nonsurgical specialists including plastic surgeons, dermatologists, gynecologists, oral surgeons, and otolaryngologists in settings ranging from hospital operating rooms to physicians' offices. The method is applied and promoted as an easy-to-learn technique that is suited as an outpatient procedure. Although major complications seem to be rare, there are definite risks, including death at a rate of 1/5,000 procedures. Major risk factors are insufficient hygiene standards, multiliter wetting solution infiltration, megavolume aspiration, multiple cosmetic procedures in one setting, sedative and anesthetic drug hangover threatening ventilation, permissive postoperative discharge, and mistakes in patient selection. When major complications occur, office-based practitioners may refer patients to hospital emergency departments, where medical personnel unfamiliar with this procedure may underestimate the risk of major complications.

Adult↗
PubMed2002

[Intravital microscopic analysis of perfusion, leukocyte-endothelial cell interaction and neovascularization after burns: an in vivo study of SKH-1/hr hairless mice]].

Breakdown of skin microcirculation is supposed to play a key role in pathophysiology of burn injury. The aim of the study was to develop a burn model, which allows repetitive quantitative in vivo analysis of microcirculation after burn injury with special focus on leukocyte endothelium interaction over a longer period of time. Male hairless mice (SKH-1/hr) were used. Deep partial thickness burns were inflicted in no-touch-technique to the ears. Intravital fluorescent microscopy in combination with FITC-dextran as a plasma-marker was used to assess standard microcirculatory parameters. Leukocytes were stained with rhodamine-6-G to study their interaction with the endothelium.

Animals↗
PubMed2001

Protegrin-1 enhances bacterial killing in thermally injured skin.

OBJECTIVE: Septic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DESIGN: For thein vitroexperiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60 degrees C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SETTING: University of Michigan research laboratory. SUBJECTS: Adult, male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. MEASUREMENTS AND MAIN RESULTS: In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. CONCLUSIONS: This study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Administration, Topical↗
PubMed2001

Feasibility of biolistic gene therapy in burns.

Skin is an especially attractive target for genetic manipulation because it is readily accessible and easily monitored for both the presence and the expression of inserted genes. This study was designed to assess the feasibility of particle mediated gene transfer to burned skin and to compare the transfection efficiency, anatomic distribution, and duration of transgene expression achievable in normal versus burned skin. Two days following scald injury of varying depths in 60 degrees C water (10 s: superficial partial; 20 s: deep partial; 40 s: full thickness) reporter gene (beta-galactosidase) constructs were delivered using a gene gun at various helium pressures (200-600 psi) to normal and burned skin. A time course study was performed to examine the kinetics of transgene expression. Animals received a superficial partial thickness burn and were sacrificed 12 h, 1, 3, 5, 7, 14, or 21 days after gene transfer. India Ink injection and immunohistochemistry were used to assess the depth of the scald injury. Transfection efficiency was measured in skin homogenates 24 h after gene transfer by morphometric and chemoluminescent assays. We found that the extent of tissue damage was directly related to the duration of heat source exposure. Reporter gene activity was significantly higher in superficial partial thickness burns compared to normal controls and gradually declined with increasing tissue injury. No activity was seen in the full thickness burn group. Beta-galactosidase activity reached a maximum level 12 h after gene transfer in both normal and superficial partial thickness burned skin with no levels seen after 5 days post-transfection. These findings indicate that particle-mediated gene transfer in thermally injured skin is feasible and may provide a means of introducing biologic agents into injured tissue capable of enhancing bacterial clearance and improving wound healing.

Animals↗
PubMed2000

Lipopolysaccharide-binding protein accelerates and augments Escherichia coli phagocytosis by alveolar macrophages.

BACKGROUND: The first step in bacterial clearance by leukocytes is attachment and phagocytosis. Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge. We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14. MATERIALS AND METHODS: Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E. coli-FITC (Ec-F) BioParticle challenge. Phagocytosed bacteria were assayed by fluorescence measurement. A time course study was also performed. RESULTS: LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion. Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min. Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis. CONCLUSION: These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner. This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment. We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor.

Acute-Phase Proteins↗
PubMed2000

Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4.

Lipopolysaccharide (LPS) binding protein (LBP) is a key serum factor that mediates LPS activation of mononuclear cells. In the presence of LBP, 1/1,000 the concentration of LPS is sufficient to activate peripheral blood monocytes. Previous studies with Kupffer cells have shown a variable effect of serum on LPS activation of these cells and led to the conclusion that, unlike extrahepatic mononuclear cells, Kupffer cells do not respond to LPS in an LBP-dependent fashion. Because there are multiple components in serum other than LBP that might affect LPS activation, these reports with serum are difficult to interpret. To investigate the specific role of LBP in LPS activation of Kupffer cells, we produced a functional recombinant rat LBP using a baculovirus expression system, which we used to selectively examine the role of LBP's on Kupffer-cell function. Isolated Kupffer cells exposed to increasing concentrations of LPS (0, 1, 10 ng/mL) showed a dose-dependent increase in TNF-alpha production, which was augmented and accelerated by the presence of LBP. The effects of LBP on Kupffer cell activation by LPS are dependent on a functional Toll-like receptor 4 (Tlr 4) because Kupffer cells from C3H/HeJ mice failed to respond to LPS in the presence of LBP. LBP plays an important role in mediating Kupffer cell activation by LPS, and these effects are dependent on the presence of functioning Tlr 4.

Acute-Phase Proteins↗