IgE blockade with omalizumab reduces pruritus related to immune checkpoint inhibitors and anti-HER2 therapies.

BACKGROUND: Immunoglobulin E (IgE) blockade with omalizumab has demonstrated clinical benefit in pruritus-associated dermatoses (e.g. atopic dermatitis, bullous pemphigoid, urticaria). In oncology, pruritus-associated cutaneous adverse events (paCAEs) are frequent with immune checkpoint inhibitors (CPIs) and targeted anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we sought to evaluate the efficacy and safety of IgE blockade with omalizumab in cancer patients with refractory paCAEs related to CPIs and anti-HER2 agents. PATIENTS AND METHODS: Patients included in this multicenter retrospective analysis received monthly subcutaneous injections of omalizumab for CPI or anti-HER2 therapy-related grade 2/3 pruritus that was refractory to topical corticosteroids plus at least one additional systemic intervention. To assess clinical response to omalizumab, we used the Common Terminology Criteria for Adverse Events version 5.0. The primary endpoint was defined as reduction in the severity of paCAEs to grade 1/0. RESULTS: A total of 34 patients (50% female, median age 67.5 years) received omalizumab for cancer therapy-related paCAEs (71% CPIs; 29% anti-HER2). All had solid tumors (29% breast, 29% genitourinary, 15% lung, 26% other), and most (n = 18, 64%) presented with an urticarial phenotype. In total 28 of 34 (82%) patients responded to omalizumab. The proportion of patients receiving oral corticosteroids as supportive treatment for management of paCAEs decreased with IgE blockade, from 50% to 9% (P < 0.001). Ten of 32 (31%) patients had interruption of oncologic therapy due to skin toxicity; four of six (67%) were successfully rechallenged following omalizumab. There were no reports of anaphylaxis or hypersensitivity reactions related to omalizumab. CONCLUSIONS: IgE blockade with omalizumab demonstrated clinical efficacy and was well tolerated in cancer patients with pruritus related to CPIs and anti-HER2 therapies.

How acoustic cavitation can improve adhesion.

In general, ultrasound is commonly used at low power level for non-destructive testing (NDT) and detection of delaminations in adhesive bonded structures. The present paper instead presents an approach where power ultrasound is used to improve interface formation prior to the bonding process and to ensure the quality of adhesive bonds by using acoustic cavitation in the liquid adhesive. Results from high-speed videos, rheological and thermal measurements and destructive testing of adhesive bonds with contaminated surfaces are presented and discussed. Power ultrasound can be used in general to improve adhesion and significantly to improve contamination tolerance and robustness of adhesive bonding processes.

Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a natural inhibitor of metalloproteinases involved in matrix degradation and ectodomain shedding of many cell-surface proteins, including death receptors and/or their ligands. In the present study, we examined the role of TIMP-3 in Fas-mediated neuronal cell death following cerebral ischemia, using both gene deletion and pharmacological approaches. In culture, exposure of primary cortical neurons to 2 h of oxygen-glucose deprivation (OGD) resulted in delayed neuronal cell death that was dependent on activation of the death receptor, Fas. Cortical cultures derived from timp-3(-/-) mice displayed partial resistance against OGD-induced neuronal cell death and also displayed increased shedding of Fas ligand (FasL) into the culture media, compared to wild-type control cultures. Both the increased neuroprotection and increased FasL shedding in timp-3(-/-) cultures were reversed by addition of exogenous metalloproteinase inhibitors, recombinant TIMP-3 or GM6001. In vivo, timp-3(-/-) mice showed marked resistance to a brief (30 min) middle cerebral artery occlusion (MCAO), but were not protected against more severe lesions induced by 90 min of MCAO. These studies demonstrate that TIMP-3 facilitates Fas-mediated neuronal cell death following OGD and plays a pro-apoptotic role in mild cerebral ischemia.

Vulnerability of mouse cortical neurons to doxorubicin-induced apoptosis is strain-dependent and is correlated with mRNAs encoding Fas, Fas-Ligand, and metalloproteinases.

Cell surface death receptor-mediated neuronal apoptosis, which is a critical component of neurodegeneration, is modulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Doxorubicin (Dox) induces neuronal death by the activation of death receptor pathways. Recently, we demonstrated that Dox-induced neuronal apoptosis is regulated by the balance of MMP-3 and TIMP-3 in rat cortical cultures. Inbred mouse strains exhibit differential susceptibility to cell death stimuli in vivo. Prior to employing transgenic approaches to further investigate the roles of TIMP-3 and MMP-3 in neuronal death, we examined whether inbred mice display strain-dependent vulnerability to Dox. We induced neuronal apoptosis with Dox in primary neuronal cultures established from cerebral cortices of embryonic day 15 C57BL/10 or C57BL/6 mice. Using fluorescence activated cell sorting for neurons, we found that C57BL/6 cortical cultures exhibit a 28% greater neuronal death following Dox treatment than C57BL/10. Real-time PCR of unstimulated cultures revealed that C57BL/10 cortical cultures have reduced basal mRNA levels encoding the pro-apoptotic proteins: Fas, FasL, and TIMP-3, but increased levels of the anti-apoptotic molecule MMP-3 as compared to C57BL/6. Furthermore, C57BL/10 cultures treated with Dox displayed an enhanced induction of mRNA transcripts that encode anti-apoptotic MMPs. These results show that C57BL/10 cortical cultures are more resistant to death receptor-mediated apoptotic cell death as compared to C57BL/6, and suggest that this difference is related to Fas, FasL, and MMP expression. Strain-dependent differences in response to apoptotic stimuli may be an important consideration for developing transgenic models of neurodegeneration.

Tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal sensitivity to doxorubicin-induced apoptosis.

Metalloproteinase activity at the cell surface influences cellular sensitivity to extrinsic death vs. survival signals in a variety of cell types, through proteolytic shedding of cell surface signalling molecules. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a unique natural metalloproteinase inhibitor that plays a pro-apoptotic role through its ability to inhibit metalloproteinases that proteolytically cleave death receptors and their ligands from the cell surface. To study the convergence of metalloproteinase activity and death receptor signalling in neurons, we established an in vitro model of neuronal apoptosis utilizing the chemotherapeutic drug, doxorubicin (Dox). Primary cultures established from embryonic rat cerebral cortices displayed robust and selective neuronal apoptosis in response to Dox, an effect that was dependent on the activation of the death receptor, Fas. We demonstrate that both TIMP-3 and matrix metalloproteinase-3 (MMP-3) are constitutively expressed by primary cortical neurons in culture, and selectively modulated Fas-mediated neuronal apoptosis induced by Dox. Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced neuronal death, whereas addition of active MMP-3 markedly attenuated apoptosis and diminished Fas-Fas ligand interaction at the cell surface. These observations implicate a physiological role for the balance of TIMP-3 and MMP-3 activity at the neuronal surface in regulating death receptor sensitivity. The convergence of metalloproteinase activity and death receptor signalling at the cell surface may influence neuronal cell death vs. survival decisions.

Cell-density-regulated chemotactic responsiveness of keratinocytes in vitro.

Keratinocytes represent the main constituents of the epidermis and have been found to play a regulatory role in a variety of inflammatory skin diseases. The functional activity of keratinocytes is highly heterogeneous, and depends on the cell localization in the epidermal architecture, and the maturation or differentiation state of the cells. Spontaneously proliferating HaCaT cells, showing several similarities to basal epidermal keratinocytes, were found to respond to external chemoattractants, including the chemokines RANTES (regulated on activation normal T cell expressed and secreted) and interleukin-8 and the mu-opioid agonist DAMGO ([d-ala2, N-Me-Phe4, Gly-ol5]enkephalin) in migration assays. The chemotactic responsiveness was highly dependent on the cell density of the monolayer, with greatest chemotactic activity at the highest cell density. Whereas RANTES was found to be the most potent chemoattractant, constitutive RANTES production was also detected in the HaCaT cultures. We found an inverse correlation between constitutive RANTES production and chemotactic responsiveness toward external RANTES, suggesting a possible functional down-modulation of the RANTES receptors, CC chemokine receptor 1 and CC chemokine receptor 5, during culture. Results from confocal laser scanning microscopy showed reduced CC chemokine receptor 1, but not CC chemokine receptor 5, expression by HaCaT cells at low cell densities, which was abolished in the presence of neutralizing antibodies against RANTES. The total CC chemokine receptor 1 pool (surface and intracellular receptors), however, showed no significant change during in vitro culture. Chemotactic responsiveness toward RANTES was directly correlated with the level of CC chemokine receptor 1 surface expression. Taken together these results show that with keratinocyte proliferation and the progressive increase in cell density there are dramatic alterations in keratinocyte function.

The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1.

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)

Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors.

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents.

Opioids, opioid receptors, and the immune response.

It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.

Should an angiotensin-converting enzyme inhibitor be standard therapy for patients with atherosclerotic disease?

Angiotensin-converting enzyme (ACE) inhibitors appear to possess unique cardioprotective benefits, even when used in patients without high blood pressure or left ventricular dysfunction (the traditional indications for ACE inhibitor therapy). The ACE inhibitors improve endothelial function and regress both left ventricular hypertrophy and arterial mass better than other antihypertensive agents that lower blood pressure equally as well. These agents promote collateral vessel development and improve prognosis in patients who have had a coronary revascularization procedure (i.e., percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery). Insulin resistance, present not only in type 2 diabetes but also commonly in patients with hypertension or coronary artery disease, or both, sensitizes the vasculature to the trophic effects of angiotensin II and aldosterone. This may partly explain the improvement in prognosis noted when patients who have atherosclerosis or diabetes are treated with an ACE inhibitor. Therapy with ACE inhibitors has also been shown, in two large, randomized trials, to reduce the incidence of new-onset type 2 diabetes through largely unknown mechanisms. The ACE inhibitors are safe, well tolerated and affordable medications. The data suggest that most people with atherosclerosis should be considered candidates for ACE inhibitor therapy, unless they are intolerant to the medication, or have systolic blood pressures consistently <100 mm Hg. Patients who show evidence of insulin resistance (with or without overt type 2 diabetes) should also be considered as candidates for prophylactic ACE inhibitor therapy. Although angiotensin receptor blockers should not be considered equivalent to ACE inhibitors for this indication, they may be a reasonable alternative for patients intolerant of ACE inhibitors.

A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells.

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro.

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

The natural history of cavernous malformations: a prospective study of 68 patients

OBJECTIVE: Cavernous malformations are angiographically occult cerebrovascular malformations found in approximately 0.5% of the population. To help further understand the natural history of these lesions, we prospectively followed 68 patients harboring cavernous malformations. METHODS: The 68 patients in this study were all diagnosed radiographically (67 patients) or surgically (1 patient) and were entered into a patient database. Age, sex, clinical symptoms, seizure frequency, focal neurological deficits, and presence or absence of extralesional hemorrhage were all recorded at presentation. Patients were then followed prospectively to determine the rate of hemorrhage and new-onset seizures. RESULTS: The mean follow-up per patient was 5.2 years, and the total follow-up was 352.9 patient-years. There was an average of 3.4 lesions per patient. Thirteen of the patients (19%) had familial cavernous malformations. Patients with familial disease were more likely to have multiple lesions than patients with sporadic disease (85% versus 25%, respectively [P = 0.001]). Initial presentation included headache (65%), seizures (49%), and focal neurological deficit (46%). Eleven symptomatic, radiologically proven, extralesional hemorrhages occurred during the 352.9 patient-years of follow-up for an overall hemorrhage rate of 3.1% per patient-year. Female patients had a significantly higher prospective hemorrhage rate (4.2% per patient-year versus 0.9% per patient-year [P = 0.04]). A history of hemorrhage at presentation was not a risk factor for subsequent hemorrhage during follow-up. The rate of new-onset seizures was 2.4% per patient-year. CONCLUSION: The clinical presentation and prospective hemorrhage rate reported here agree well with findings of other prospective studies. This information, combined with our new-onset seizure rate, should aid clinicians caring for patients with cavernous malformations.

Courses involving complementary and alternative medicine at US medical schools.

CONTEXT: With the public's increasing use of complementary and alternative medicine, medical schools must consider the challenge of educating physicians about these therapies. OBJECTIVES: To document the prevalence, scope, and diversity of medical school education in complementary and alternative therapy topics and to obtain information about the organizational and academic features of these courses. DESIGN: Mail survey and follow-up letter and telephone survey conducted in 1997-1998. PARTICIPANTS: Academic or curriculum deans and faculty at each of the 125 US medical schools. MAIN OUTCOME MEASURES: Courses taught at US medical schools and administrative and educational characteristics of these courses. RESULTS: Replies were received from 117 (94%) of the 125 US medical schools. Of schools that replied, 75 (64%) reported offering elective courses in complementary or alternative medicine or including these topics in required courses. Of the 123 courses reported, 84 (68%) were stand-alone electives, 38 (31%) were part of required courses, and one (1%) was part of an elective. Thirty-eight courses (31%) were offered by departments of family practice and 14 (11%) by departments of medicine or internal medicine. Educational formats included lectures, practitioner lecture and/or demonstration, and patient presentations. Common topics included chiropractic, acupuncture, homeopathy, herbal therapies, and mind-body techniques. CONCLUSIONS: There is tremendous heterogeneity and diversity in content, format, and requirements among courses in complementary and alternative medicine at US medical schools.

An immune cell-selective interleukin 4 agonist.

Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4Ralpha/IL-2Rgamma (class I IL-4R), whereas endothelial cells express another type, IL-4Ralpha/IL-13Ralpha (class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2Rgamma. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/R121E retained physical interaction with IL-2Rgamma but not IL-13Ralpha; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activities in vitro that suggest utility in the treatment of certain autoimmune diseases.

Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay.

The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.