Monocytosis in primary care and risk of haematological malignancies.

Monocytosis (≥0.5 × 109 /L in peripheral blood) is the hallmark of chronic myelomonocytic leukaemia (CMML) but may be present in a spectrum of diseases including other haematological malignancies. In the primary care sector, monocytosis is a relatively common finding, but its predictive value for haematological malignancy is unknown. We included 663 184 adult primary care patients from the greater Copenhagen area with one or more differential cell counts registered between 2000 and 2016 and followed them in the extensive nationwide Danish health data registers for 3 years after blood sampling. We used logistic regression to model the risk of haematological malignancy and death following monocytosis. Monocytosis was associated with an increased risk of all types of haematological malignancy with the greatest relative risk increase observed in CMML with an OR of 105.22 (95% confidence interval: 38.27-289.30). Sustained monocytosis (at least two requisitions in 3 months) further increased CMML risk, although the diagnosis was still very rare, that is, observed in only 0.1% of these individuals. Outside the haematological setting, the absolute risk of haematological malignancy associated with monocytosis is low and haematological malignancy should mainly be suspected when monocytosis is sustained or the clinical presentation raises suspicion of malignancy.

Targeting the CCL2-CCR2 axis for atheroprotection.

Decades of research have established atherosclerosis as an inflammatory disease. Only recently though, clinical trials provided proof-of-concept evidence for the efficacy of anti-inflammatory strategies with respect to cardiovascular events, thus offering a new paradigm for lowering residual vascular risk. Efforts to target the inflammasome-interleukin-1ß-interleukin-6 pathway have been highly successful, but inter-individual variations in drug response, a lack of reduction in all-cause mortality, and a higher rate of infections also highlight the need for a second generation of anti-inflammatory agents targeting atherosclerosis-specific immune mechanisms while minimizing systemic side effects. CC-motif chemokine ligand 2/monocyte-chemoattractant protein-1 (CCL2/MCP-1) orchestrates inflammatory monocyte trafficking between the bone marrow, circulation, and atherosclerotic plaques by binding to its cognate receptor CCR2. Adding to a strong body of data from experimental atherosclerosis models, a coherent series of recent large-scale genetic and observational epidemiological studies along with data from human atherosclerotic plaques highlight the relevance and therapeutic potential of the CCL2-CCR2 axis in human atherosclerosis. Here, we summarize experimental and human data pinpointing the CCL2-CCR2 pathway as an emerging drug target in cardiovascular disease. Furthermore, we contextualize previous efforts to interfere with this pathway, scrutinize approaches of ligand targeting vs. receptor targeting, and discuss possible pathway-intrinsic opportunities and challenges related to pharmacological targeting of the CCL2-CCR2 axis in human atherosclerotic disease.

Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus.

Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte-macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.

Successful treatment of pyoderma gangrenosum with granulocyte and monocyte adsorption apheresis.

Pyoderma gangrenosum is a neutrophilic dermatosis clinically characterised by the presence of painful skin ulcerations with erythematous and undetermined borders and histologically by the presence of neutrophilic infiltrates in the dermis. Granulocyte and monocyte adsorption apheresis, also called granulocytapheresis, is a therapeutic strategy for extracorporeal immunomodulation that selectively removes activated granulocytes and monocytes/macrophages from the peripheral blood. Here, we report a case of a 73-year-old patient affected by a severe form of pyoderma gangrenosum presenting with multiple painful ulcers and pustules on his trunk and extremities. The disease was resistant to high doses of methylprednisolone and methotrexate and successfully treated by granulocyte and monocyte adsorption apheresis. To the best of our knowledge, this is the first report on the efficacy of granulocyte and monocyte adsorption apheresis in pyoderma gangrenosum in Europe.

Rosuvastatin treatment reduces markers of monocyte activation in HIV-infected subjects on antiretroviral therapy.

BACKGROUND: Statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have anti-inflammatory effects that are independent of their lipid-lowering properties. Despite suppressive antiretroviral therapy (ART), elevated levels of immune activation and inflammation often persist. METHODS: The Stopping Atherosclerosis and Treating Unhealthy Bone With Rosuvastatin in HIV (SATURN-HIV) trial is a randomized, double-blind, placebo-controlled study, designed to investigate the effects of rosuvastatin (10 mg/daily) on markers of cardiovascular disease risk in ART-treated human immunodeficiency virus (HIV)-infected subjects. A preplanned analysis was to assess changes in markers of immune activation at week 24. Subjects with low-density lipoprotein cholesterol <130 mg/dL and heightened immune activation (%CD8(+)CD38(+)HLA-DR(+) ≥19%, or plasma high-sensitivity C-reactive protein ≥2 mg/L) were randomized to receive rosuvastatin or placebo. We measured plasma (soluble CD14 and CD163) and cellular markers of monocyte activation (proportions of monocyte subsets and tissue factor expression) and T-cell activation (expression of CD38, HLA-DR, and PD1). RESULTS: After 24 weeks of rosuvastatin, we found significant decreases in plasma levels of soluble CD14 (-13.4% vs 1.2%, P = .002) and in proportions of tissue factor-positive patrolling (CD14(Dim)CD16(+)) monocytes (-38.8% vs -11.9%, P = .04) in rosuvastatin-treated vs placebo-treated subjects. These findings were independent of the lipid-lowering effect and the use of protease inhibitors. Rosuvastatin did not lead to any changes in levels of T-cell activation. CONCLUSIONS: Rosuvastatin treatment effectively lowered markers of monocyte activation in HIV-infected subjects on antiretroviral therapy. CLINICAL TRIALS REGISTRATION: NCT01218802.

Extra-virgin olive oil consumption reduces the age-related decrease in HDL and paraoxonase 1 anti-inflammatory activities.

Paraoxonase 1 (PON1) is associated with HDL and modulates the antioxidant and anti-inflammatory role of HDL. The goals of the present study were to investigate the effect of ageing and the role of PON1 on the anti-inflammatory activity of HDL, and to determine whether extra-virgin olive oil (EVOO) consumption could improve the atheroprotective activity of HDL. HDL and PON1 were isolated from the plasma of ten young (Y-HDL and Y-PON1) and ten elderly (E-HDL and E-PON1) healthy volunteers before and after 12 weeks of EVOO consumption. Inflammation was assessed by measuring intracellular adhesion molecule 1 (ICAM-1) expression. THP-1 (human acute monocytic leukaemia cell line) monocyte chemotaxis was measured using a Boyden chamber. Oxidative damage to HDL was assessed by measuring conjugated diene formation and changes in electrophoretic migration. Y-HDL had more anti-inflammatory activity than E-HDL. The conjugated diene content and the electrophoretic mobility of E-HDL were higher than those of Y-HDL. Y-PON1 had significant anti-inflammatory activity, reducing ICAM-1 expression by 32·64 (SD 2·63)%, while E-PON1 had no significant effect. THP-1 chemotaxis measurements confirmed the ICAM-1 expression results. The 12 weeks of EVOO consumption significantly increased the anti-inflammatory activities of both HDL and PON1. The anti-inflammatory activity of HDL was modulated by PON1 and was lower in the elderly volunteers. EVOO consumption increased the anti-inflammatory effect of HDL and reduced the age-related decrease in anti-atherogenic activity.

Rationale and efficacy of interleukin-1 targeting in Erdheim-Chester disease.

Erdheim-Chester disease (ECD) pathophysiology remains largely unknown. Its treatment is not codified and usually disappointing. Interferon (IFN)-α therapy lacks efficacy for some life-threatening manifestations and has a poor tolerance profile. Because interleukin (IL)-1Ra synthesis is naturally induced after stimulation by IFN-α, we hypothesized that recombinant IL-1Ra (anakinra) might have some efficacy in ECD. We treated 2 patients who had poor tolerance or contraindication to IFN-α with anakinra as a rescue therapy and measured their serum C-reactive protein, IL-1ß, IL-6, and monocytic membranous IL-1α (mIL-1α) levels before, under, and after therapy. Another untreated ECD patient and 5 healthy subjects were enrolled as controls. After treatment, fever and bone pains rapidly disappeared in both patients, as well as eyelid involvement in one patient. In addition, retroperitoneal fibrosis completely or partially regressed, and C-reactive protein, IL-6, and mIL-1α levels decreased to within the normal and control range. Beside injection-site reactions, no adverse event was reported. Therefore, our results support a central role of the IL-1 network, which seemed to be overstimulated in ECD. Its specific blockade using anakinra thereby opens new pathophysiology and therapeutic perspectives in ECD.

Increased activation of the transcription factor c-Jun by MAP kinases in monocytes of multiple trauma patients is associated with adverse outcome and mass transfusion.

BACKGROUND: Post-traumatic dysfunction of the immune system is a major source of morbidity and mortality in patients with multiple trauma. The underlying intracellular mechanisms are still incompletely understood. Previous mRNA expression studies in monocytes suggested an involvement of the MAP kinases p38 and JNK and of the transcription factor c-Jun. Therefore, it was the aim of this study to elucidate whether alterations in the protein expression p38 MAPK, JNK, and c-Jun could be linked to PRBC substitution and survival. MATERIALS AND METHODS: Thirty-seven patients with blunt multiple injuries and an ISS > 16 points were enrolled in our study. Blood was drawn on admission and 6, 12, 24, 48, and 72 h after the traumatic event. Monocytes were isolated immediately after sample collection and nuclear protein was extracted and phosphoprotein concentrations were measured. The resulting data were statistically analyzed. RESULTS: An increased activation of MAP kinases and c-Jun could be shown in patients who died from their injuries. Additionally, patients who received PRBC substitution ≥10 units exhibited increased expression of activated MAP kinases and c-Jun. CONCLUSIONS: We present a serial, sequential investigation in human monocytes of major trauma patients evaluating the activation of p38 MAPK, JNK and c-Jun in the post-traumatic period. We show that death after trauma and massive PRBC substitution are associated with activation of this pathway. The p38 MAPK, JNK, and c-Jun have well established proinflammatory properties. Therefore, it appears likely that this pathway is involved in the systemic hyperinflammatory states seen after massive PRBC transfusion and multiple trauma.

Granulocyte and monocyte adsorptive apheresis for pyoderma gangrenosum.

Pyoderma gangrenosum (PG), a chronic aseptic inflammatory skin disease characterized by skin ulcers with elevated and undermined borders, is resistant to conventional therapies. PG is elicited by activated neutrophils and macrophages and is often associated with systemic diseases such as inflammatory bowel disease, rheumatoid arthritis, aortitis syndrome, and hematopoietic disorders. This single-center study assessed the efficacy and safety of selectively depleting myeloid-lineage leukocytes in patients with PG. Patients with PG, aged 20 or over, received 5 or 10 treatment sessions of granulocyte and monocyte adsorption apheresis (GMA), once or twice a week. Treatment efficacy was assessed based on the rate of skin ulcer reduction, the visual analog scale of pain, and the physician's global assessment of the skin lesions. A complete response (CR) was obtained in eight patients, a nearly complete response (nCR) in three patients, and a partial response (PR) in two patients. In four of the other six, the disease remained stable (SD) and in two we observed disease progression (PD). No severe adverse events were recorded. Our results suggest that GMA is a useful and safe treatment modality for PG.

PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

Introduction: Circulating monocytes are main source for tumor-associated macrophages (TAMs) that control tumor growth, angiogenesis, metastasis and therapy resistance. We raised the questions how monocyte programming is affected by growing tumors localized in colon and rectal sections, and how treatment onsets affect monocyte programming in the circulation. Methods: Patients with rectal cancer and colon cancer were enrolled in the study. Peripheral blood monocytes were characterized by phenotypic analysis using flow cytometry, by transcriptomic analysis using RNA sequencing and by gene expression analysis using real-time RT-PCR. Phenotypic analysis was performed with IF/confocal microscopy. Spatial transcriptomic analysis was applied using GeoMX DSP-NGS. Results: In patients with rectal cancer, increased amount of CCR2+ monocytes was indicative for the absence of both lymphatic and hematogenous metastasis. In contrast, in patients with colon cancer CD163+ monocytes were indicative for LN metastasis. NGS analysis identified tumor-specific transcriptional programming of monocytes in all CRC patients compared to healthy individuals. The key transcriptional difference between monocytes of patients with colon and rectal cancer was increased expression of PFKFB3, activator of glycolysis that is currently considered as therapy target for major solid cancers. PFKFB3-expressing monocyte-derived macrophages massively infiltrated tumor in colon. Nanostring technology identified correlation of PFKFB3 with amount and tumor-promoting properties of TAMs in colon but not in rectal cancer. PFKFB3 was indicative for tumor relapse specifically in colon cancer. Discussion: Our findings provide essential argument towards CRC definition to cover two clinically distinct cancers - colon cancer and rectal cancer, that differentially interact with innate immunity.

Mucosal healing of esophageal involvement of Crohn's disease with granulocyte/monocyte adsorption.

We report a case of a 16-year-old male who suffered from Crohn's disease (CD) with esophageal involvement, showing remarkable improvement with granulocyte/monocyte adsorption (GMA). The patient had been diagnosed as ileocolic CD and was treated with 5-aminosalicylate. He was admitted to our hospital with symptoms of fever, diarrhea, and odynophagia. Endoscopic examinations revealed that the exacerbation of ileocolic ulcers, and advent of ulcers in esophagus. Because of the patient's refusal to receive corticosteroids, immunomodulators, or biologics, he underwent GMA twice a week. After 10 sessions of GMA, he entered remission with significant decrease in clinical activity. In addition, endoscopic examinations showed remarkable improvement of ileocolic ulcers and disappearance of esophageal lesions. No adverse events were observed. GMA could be effective for manifestations of CD in gastrointestinal tract other than ileum or colon.

IL-6 modulation for COVID-19: the right patients at the right time?

The ongoing pandemic caused by the novel coronavirus SARS-CoV-2 has disrupted the global economy and strained healthcare systems to their limits. After the virus first emerged in late 2019, the first intervention that demonstrated significant reductions in mortality for severe COVID-19 in large-scale trials was corticosteroids. Additional options that may reduce the burden on the healthcare system by reducing the number of patients requiring intensive care unit support are desperately needed, yet no therapy has conclusively established benefit in randomized studies for the management of moderate or mild cases of disease. Severe COVID-19 disease is characterized by a respiratory distress syndrome accompanied by elevated levels of several systemic cytokines, in a profile that shares several features with known inflammatory pathologies such as hemophagocytic lymphohistiocytosis and cytokine release syndrome secondary to chimeric antigen receptor (CAR) T cell therapy. Based on these observations, modulation of inflammatory cytokines, particularly interleukin (IL)-6, was proposed as a strategy to mitigate severe disease. Despite encouraging recoveries with anti-IL-6 agents, especially tocilizumab from single-arm studies, early randomized trials returned mixed results in terms of clinical benefit with these interventions. Later, larger trials such as RECOVERY and REMAP-CAP, however, are establishing anti-IL-6 in combination with steroids as a potential option for hypoxic patients with evidence of hyperinflammation. We propose that a positive feedback loop primarily mediated by macrophages and monocytes initiates the inflammatory cascade in severe COVID-19, and thus optimal benefit with anti-IL-6 therapies may require intervention during a finite window of opportunity at the outset of hyperinflammation but before fulminant disease causes irreversible tissue damage-as defined clinically by C reactive protein levels higher than 75 mg/L.

Recombinant Alkaline Phosphatase Prevents Acute on Chronic Liver Failure.

The lipopolysaccharide (LPS)- toll-like receptor-4 (TLR4) pathway plays an important role in liver failure. Recombinant alkaline phosphatase (recAP) deactivates LPS. The aim of this study was to determine whether recAP prevents the progression of acute and acute-on-chronic liver failure (ACLF). Eight groups of rats were studied 4-weeks after sham surgery or bile duct ligation and were injected with saline or LPS to mimic ACLF. Acute liver failure was induced with Galactosamine-LPS and in both models animals were treated with recAP prior to LPS administration. In the ACLF model, the severity of liver dysfunction and brain edema was attenuated by recAP, associated with reduction in cytokines, chemokines, liver cell death, and brain water. The activity of LPS was reduced by recAP. The treatment was not effective in acute liver failure. Hepatic TLR4 expression was reduced by recAP in ACLF but not acute liver failure. Increased sensitivity to endotoxins in cirrhosis is associated with upregulation of hepatic TLR4, which explains susceptibility to development of ACLF whereas acute liver failure is likely due to direct hepatoxicity. RecAP prevents multiple organ injury by reducing receptor expression and is a potential novel treatment option for prevention of ACLF but not acute liver failure.

Recipient Hepatic Tumor-Associated Immunologic Infiltrates Predict Outcomes After Liver Transplantation for Hepatocellular Carcinoma.

BACKGROUND Transplantation of the liver entails a state of altered recipient immunologic competence. There are only scarce data concerning the impact of host immunologic factors on the outcome of liver transplant recipients in the context of hepatocellular carcinoma (HCC). MATERIAL AND METHODS Our study focused on evaluating the presence of tumor necrosis and frequency levels of angiopoietins and monocytes/macrophages subtypes in the host liver prior to liver transplantation (LTX) and their association with recurrence, graft rejection, survival, and clinical prognosis after LTX. Formation of tumor necrosis and tissue densities of angiopoietins and cellular immunologic infiltrates - CD68⁺ and CD163⁺ macrophages (TAMs) and TIE2-expressing monocytes (TEMs) - were quantified in recipient HCC specimens. The densities were then matched with clinicopathologic variables and patient survival after LTX (n=88). Some patients were treated prior to LTX by neoadjuvant transarterial chemoembolization (TACE, n=55). RESULTS Recipient hepatic infiltration with TEMs and CD68⁺ TAMs was associated with decreased 1-, 3-, and 5-year survival, as well as metastatic and recurrent HCC after LTX (all p<0.05). TEMs and infiltrating monocytes/macrophages were associated with angiopoietin expression, metastatic, and recurrent HCC (all p<0.05). Furthermore, hepatic angiopoietin-2 expression was associated with graft rejection after LTX (p<0.05). After TACE and LTX, formation of tumor necrosis was associated with an increased presence of monocytes/macrophages and a reduced incidence of recurrent HCC in the graft (all p<0.05). CONCLUSIONS Infiltrating monocytes/macrophages subsets and related angiopoietin axis are associated with worse survival, tumor recurrence, and clinical outcome after LTX for HCC.

Refractory Ulcerative Colitis Improved by Scheduled Combination Therapy of Vedolizumab and Granulocyte and Monocyte Adsorptive Apheresis.

Granulocyte and monocyte adsorptive apheresis (GMA) is occasionally introduced as an alternative combination therapy after loss of response to biologics in ulcerative colitis (UC) patients. However, there have been no reports of the concomitant use of vedolizumab (VDZ) and GMA for the initial induction of UC. A 20-year-old man with refractory UC was admitted for recrudescence. VDZ monotherapy had previously been introduced but was ineffective. Therefore, he received scheduled combination of VDZ and GMA and achieved clinical remission. The combination of two different approaches to inhibit the migration of leukocytes into the inflamed tissue led to satisfactory clinical outcomes.

Prevention of Obesity Related Diseases through Laminarin-induced targeted delivery of Bindarit.

Rationale: The developement of oral targeted therapeutics for obesity and obesity-related diseases is challenging, as these diseases involve multiple lesions distributed throughout the whole body. Herein, we report a successful stragety for targeted oral delivery of bindarit to multiple obesity-related lesions including inflamed adipose tissue, fatty liver and atherosclerotic plaques. Methods: The computer simulation from atomstic to mesoscale was first applied for designing bindarit-loaded nanoparticles (pBIN) and laminarin-modified bindarit-loaded nanoparticles (LApBIN). Then pBIN were suceesfully prepared using a dialysis procedure, and LApBIN were prepared though the interaction bewtween laminarin and pBIN. The physiochemical properties, in vitro and in vivo pharmacokinetics, oral targeting capability and in vivo efficacy of LApBIN in various obesity-related diseases were examined. Results: LApBIN were sucessfully designed and prepared. Following oral administration of LApBIN, the nanoparticles could be sucessully orally adsorbed and translocated to monocytes. Contributed by the recruitment of monocytes to multiple obesity-related lesions, LApBIN successfully delivered bindarit to these lesions, and effectively suppressed inflammation there, which exerted successful preventive effects on high-fat-diet-induced obesity, insulin resistance, fatty liver and atherosclerosis. Conclusions:This strategy could represent a promising approach to develop effective oral treatments for obesity and other metabolic diseases.

Development and validation of a monocyte activation test for the control/safety testing of an OMV-based meningococcal B vaccine.

It is imperative to ensure biological products are free of contaminating pyrogenic material prior to administration to patients. Historically the rabbit pyrogen test (RPT) was used to screen for such contamination in medicines for intravenous delivery. This test was adapted for use to screen vaccines. However, some, including meningococcal vaccines containing outer membrane vesicles, are intrinsically pyrogenic. Indeed, this is the case for Bexsero which contains relatively high levels of endotoxin and other potential pyrogens such as lipoproteins and porins. The RPT proved a difficult method for measuring the pyrogenic content of Bexsero and differences between laboratories in different countries made repeat testing at the control laboratories problematic resulting in batches being wrongly identified as unsafe. At NIBSC a monocyte activation test (MAT) was adapted and validated as an alternative. This required setting of a specification in-house and deciding on a decisional procedure using multiple donors, allowing batches equally pyrogenic or less, than those batches shown to be safe in a clinical trial, to be certified as safe. The resulting format was a reference comparison method with an upper limit of 1.8 relative pyrogen units (RPU). The batch passed if an initial four donors had a response equal to or less than 1.8 RPU, if one donor is above this limit the batch was tested in a further four donors and seven of the eight must be equal to or below 1.8 RPU. If two donors have a response greater than 1.8 the batch failed.

Monocyte-activation test to reliably measure the pyrogenic content of a vaccine: An in vitro pyrogen test to overcome in vivo limitations.

Pyrogen content is one of the critical quality attributes impacting the safety of a product, and there is an increasing need for assays that can reliably measure this attribute in vaccines. The Limulus amebocyte lysate (LAL) assay and the rabbit pyrogen test (RPT) are the canonical animal-based pyrogen tests currently used to release vaccines; however, there are several drawbacks associated with these tests when applied to Bexsero, intrinsically pyrogenic product, containing a meningococcal Outer Membrane Vesicle component. While the RPT, as applied to Bexsero at its given dilution, ensures safe vaccine, it is highly variable and prone to false positive results. On the other hand, the LAL assay although quantitative, can detect only endotoxin pyrogens and is not sufficient for monitoring the safety of Bexsero, which contains both LPS and non-endotoxin pyrogens. Being aware of these limitations of the RPT and LAL when applied to Bexsero, the Monocyte Activation Test (MAT) which is sensitive to both endotoxin and non-endotoxin based pyrogens has been developed as an alternative pyrogen test. Here, the development and the validation of a MAT assay adapted from the European pharmacopoeia for Bexsero, is described. The MAT assay is then used for monitoring the safety and consistency of Bexsero vaccines at release, providing great advantages in terms of reduced variability with respect to RPT, reduction of animal use, in line with the 3Rs principle concerning the protection of animals and faster time to market. In addition the correlation of the MAT to the RPT has been demonstrated supporting the replacement of the in vivo method and the potential application of the assay to other intrinsically pyrogenic vaccines.

Intracutaneous injection of the macrophage-activating lipopeptide-2 (MALP-2) which accelerates wound healing in mice--a phase I trial in 12 patients.

Chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, are a challenge to physicians and medical personnel and a cause of tremendous discomfort and ensuing loss of quality of life to the patients. Wound healing involves production and action of various growth factors. A novel approach, distinct from the application of single growth factors, is the administration of the macrophage stimulator macrophage-activating lipopeptide-2 (MALP-2). The rationale is based on the finding that macrophages are the main source of several growth factors required for wound healing, which are sequentially released during this process. MALP-2 has previously been shown to be effective in an established animal model with diabetic mice. The purpose of the present phase I study was to establish tolerability of MALP-2 when applied into small cutaneous wounds in human beings. Twelve patients (six females and six males; mean age 66.8 years; range 52-87 years) with different diagnoses were enrolled into the study. An artificial wound was created with a 2-mm diameter skin biopsy punch and a volume of 30 microl MALP-2 (0.125-1 microg) or vehicle control, respectively, was injected intracutaneously into the wound and closed with a water-resistant transparent adhesive. Photos were taken daily from every patient up to 6 days, and skin biopsies were performed after 1 week from six patients. We could show in the present study for the first time that MALP-2 caused a transient erythema and was tolerated without any systemic side effects up to a dose of 1 microg per wound in human beings. In healthy as well as in diabetic patients, MALP-2 induced local inflammation that faded after 48 h. The effectiveness of MALP-2 in the healing of chronic wounds in humans, e.g. in chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, could now be addressed in further studies.