Extracellular vesicles for nucleic acid delivery: progress and prospects for safe RNA-based gene therapy.

Nucleic acid-based drugs offer a potentially effective tool for treatment of a variety of diseases, including cancer, cardiovascular diseases, neurological disorders and infectious diseases. However, clinical applications are hindered by instability of RNA molecules in the circulation and lack of efficient vectors that can deliver RNAs to target tissues and into diseased target cells. Synthetic polymer and lipids as well as virus-based vectors are among the most widely explored vehicles for RNA delivery, but clinical progress has been limited as a result of issues related to toxicity, immunogenicity and low efficiency. Most recently, the discovery that extracellular vesicles (EVs) are endogenous RNA carriers, which may display better biocompatibility and higher delivery efficiency as compared with the synthetic systems, has provided a ray of hope in coping with the delivery dilemma, and EV-based gene therapy has already sparked general interest both in academia and industry. In this review, the current knowledge on EV biology and their role in cell-cell communication will be summarized. Promises of EVs as drug carriers and recent technologies on tailoring EVs' biological attributes will be included, and preclinical studies in which EVs have shown promise for therapeutic RNA delivery will be discussed.

Cardiac delivery of modified mRNA using lipid nanoparticles: Cellular targets and biodistribution after intramyocardial administration.

Despite research efforts being made towards preserving (or even regenerating) heart tissue after an ischemic event, there is a lack of resources in current clinical treatment modalities for patients with acute myocardial infarction that specifically address cardiac tissue impairment. Modified messenger RNA (modRNA) presents compelling properties that could allow new therapeutic strategies to tackle the underlying molecular pathways that ultimately lead to development of chronic heart failure. However, clinical application of modRNA for the heart is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of RNA delivery systems, which were recently approved for clinical usage in mRNA-based COVID-19 vaccines. In this study, we evaluated the potential of LNPs for cardiac delivery of modRNA. We tested how variations in C12-200 modRNA-LNP composition affect transfection levels and biodistribution after intramyocardial administration in both healthy and myocardial-infarcted mice, and determined the targeted cardiac cell types. Our data revealed that LNP-mediated modRNA delivery outperforms the current state of the art (modRNA in citrate buffer) upon intramyocardial administration in mice, with only minor differences among the formulations tested. Furthermore, we determined both in vitro and in vivo that the cardiac cells targeted by modRNA-LNPs include fibroblasts, endothelial cells and epicardial cells, suggesting that these cell types could represent targets for therapeutic interference with these LNP formulations. These outcomes may serve as a starting point for LNP development specifically for therapeutic mRNA cardiac delivery applications.

Tryptase and histamine metabolites as diagnostic indicators of indolent systemic mastocytosis without skin lesions.

BACKGROUND: Risk indicators of indolent systemic mastocytosis (ISM) in adults with clinical suspicion of ISM without accompanying skin lesions [urticaria pigmentosa (UP)] are lacking. This study aimed at creating a decision tree using clinical characteristics, serum tryptase, and the urinary histamine metabolites methylimidazole acetic acid (MIMA) and methylhistamine (MH) to select patients for bone marrow investigations to diagnose ISM. METHODS: Retrospective data analysis of all adults, in whom bone marrow investigations were performed to diagnose ISM, was carried out. RESULTS: In total, 142 patients were included. SM was absent in all 44 patients with tryptase <10 µg/l, in 45 of 98 (46%) patients with tryptase ≥10 µg/l and in 18 of 52 patients (35%) with tryptase >20 µg/l. Above 43 µg/l, all patients had ISM (n = 11). Male gender, insect venom anaphylaxis as presenting symptom, tryptase, MIMA, and MH were independent ISM predictors. If tryptase was ≥10 µg/l, the diagnostic accuracy of MIMA and MH was high (areas under the ROC curve 0.92). CONCLUSIONS: In suspected patients without UP, the ISM risk is very low (if present at all) if tryptase is <10 µg/l. If tryptase is ≥10 µg/l, this risk depends on MIMA and MH, being low if these are normal, but high if these are elevated. Male gender and insect venom anaphylaxis are additional risk indicators. We recommend refraining from bone marrow examinations in suspected patients without UP if tryptase is <10 µg/l. Our results question the reliability of the minor diagnostic World Health Organization criterion of tryptase >20 µg/l.

Influence of short term storage conditions, concentration methodsand excipients on extracellular vesicle recovery and function.

Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage. Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay. Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and -80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach.

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery.

Treatment of multifocal primary cutaneous B-cell lymphoma: a clinical follow-up study of 29 patients.

PURPOSE: Although patients with primary cutaneous B-cell lymphoma (CBCL) and localized skin lesions are generally treated with radiotherapy and have an excellent prognosis, the clinical behavior and optimal treatment of CBCL presenting with multifocal skin lesions are less well defined. In this study, we evaluated the clinical behavior of and results of treatment for multifocal CBCL in 29 patients, and we formulated therapeutic guidelines. PATIENTS AND METHODS: The study group included 16 patients with primary cutaneous follicular center-cell lymphoma (PCFCCL), eight with primary cutaneous immunocytoma (PCI), and five with primary cutaneous large B-cell lymphoma presenting on the legs (PCLBCL of the leg). RESULTS: Only one of the 24 patients with multifocal PCFCCL or PCI developed extracutaneous disease, and no patient died from lymphoma (median follow-up, 54 months). In patients with PCFCCL, treatment with either multiagent chemotherapy (nine patients) or radiotherapy directed toward all skin lesions (five patients) proved equally effective in terms of complete remission, relapse, and survival. In contrast, all five patients with PCLBCL of the leg developed extracutaneous disease, and four of the five died from systemic lymphoma, 8 to 36 months (median, 21 months) after diagnosis. CONCLUSION: The results of these preliminary studies suggest that patients with PCFCCL or PCI presenting with multifocal skin lesions have the same excellent prognosis that patients with localized PCFCCL or PCI have and that radiotherapy directed toward all skin lesions is as effective as multiagent chemotherapy. Patients with PCLBCL of the leg have a more unfavorable prognosis, particularly patients presenting with multifocal skin lesions. This last group should always be treated with multiagent chemotherapy.

Primary cutaneous marginal zone B-cell lymphoma: clinical and therapeutic features in 50 cases.

BACKGROUND: Primary cutaneous marginal zone B-cell lymphoma (PCMZL) is a low-grade B-cell lymphoma that originates in the skin, with no evidence of extracutaneous disease. Studies focusing on the optimal treatment of PCMZL have not been published thus far. We describe 50 patients with PCMZL to further characterize clinical characteristics and outcome and, in particular, to evaluate our current therapeutic approach. OBSERVATIONS: The majority of the patients (36/50 [72%]) presented with multifocal skin lesions, and 14 patients (28%) presented with solitary or localized lesions. The initial treatment of patients with solitary lesions consisted of radiotherapy or excision, whereas patients with multifocal lesions received a variety of initial treatments, most commonly radiotherapy and chlorambucil therapy. Cutaneous relapses developed in 19 (48%) of 40 patients who had complete remission and were more common in patients with multifocal disease. After a median period of follow-up of 36 months, 2 patients developed extracutaneous disease, but none of the patients died of lymphoma. CONCLUSIONS: Patients with PCMZL who have solitary lesions can be treated effectively with radiotherapy or excision. For patients with PCMZL who have multifocal lesions, chlorambucil therapy and radiotherapy are suitable therapeutic options. In case of cutaneous relapses, the beneficial effects of treatment should carefully be weighed against the potential adverse effects.

[Corticosteroids and the risk of glaucoma]. / Corticosteroïden en het risico van glaucoom.

Corticosteroids are used to treat many diseases and are prescribed by both specialists and general practitioners. One serious side effect of steroid use is glaucoma. This complication, which can cause blindness, is often only discovered at the end stage. Three patients, two women aged 20 and 32 and a man aged 28 developed glaucoma as a result of topical steroid use. It is advisable to examine patients annually if they have a family history of glaucoma and are using steroids regularly in or around the eye. Follow-up should also be considered for patients with a family history of glaucoma who are using systemic steroids regularly, and for all other patients using steroids regularly in or around the eye. All patients on steroids should consult their ophthalmologist speedily if visual symptoms occur.

Defined in situ enumeration of T6 and HLA-DR expressing epidermal Langerhans cells: morphologic and methodologic aspects.

An essential prerequisite for the in situ enumeration of epidermal Langerhans cells (LCs) is the unequivocal identification of the desired cell type. We have examined over 250 cryostat sections of normal human skin to analyze morphologic and methodologic problems underlying the quantification of epidermal LCs, defined by anti-T6 (OKT6) and anti-HLA-DR (OKIal) immunoperoxidase staining. Our findings show that OKT6 reactivity of dendritic processes in cross-sectioned epidermis yields microscopic images which are not easy to analyze objectively. The morphology that we find leads us to categorize dendritic cells into 3 arbitrary types of T6+ LC profiles. In addition we describe criteria for the assessment of OKT6 staining patterns relating to the dendritic state of epidermal LCs. Preliminary quantitative data on this issue are discussed in relation to: epidermal thickness; the thickness of skin tissue sections; and the discrepancy between the number of T6+ and HLA-DR+ LCs. We hope that the principles outlined in this report may serve to overcome potential methodologic problems with quantitation of T6+ epidermal LCs in skin sections.

Homozygous R788W point mutation in the XPF gene of a patient with xeroderma pigmentosum and late-onset neurologic disease.

The second Caucasian xeroderma pigmentosum patient (XP42RO) belonging to complementation group F (XP-F) is described. Mild ocular photophobia was present from childhood, and acute skin reactions occurred upon exposure to sunlight. Basal and squamous cell carcinomas developed after his twenty-seventh year. In his late forties progressive neurologic symptoms emerged, which included intellectual decline, mild chorea and ataxia, and marked cerebral and cerebellar atrophy. Such neurologic abnormalities are very unusual in XP-F. Similar symptoms have been described in only one of 17 other XP-F individuals. His approximately 5-fold reduced activity of nucleotide excision repair in cultured cells, combined with moderately affected cell survival and DNA replication after UV exposure, are typical of XP-F. The recent cloning of the XPF gene allowed a molecular genetic analysis of this unusual patient. XP42RO, representing the second case studied in this respect, turned out to be homozygous for a point mutation in the XPF gene, causing an R788-->W substitution in the encoded protein. Surprisingly, this mutation had also been found in one allele of the other unrelated Caucasian XP-F case. The amount of mutated XPF protein is strongly reduced in cells from XP42RO, presumably due to a conformational change. Biochemical, genetic, and clinical data all indicate the presence of considerable residual repair activity, strongly suggesting that the R788W mutation is leaky.

RET mutation screening in familial cutaneous lichen amyloidosis and in skin amyloidosis associated with multiple endocrine neoplasia.

In several families, multiple endocrine neoplasia type 2A (MEN 2A) has been found in association with cutaneous lichen amyloidosis. It has been debated, however, whether the skin amyloidosis found in MEN 2A families, localized exclusively in the interscapular area, represents the same anomaly as that found in autosomal dominant familial cutaneous lichen amyloidosis, which is more generalized. We screened two MEN 2A families with associated skin amyloidosis for germline mutations in the RET gene responsible for the MEN 2A cancer syndrome, and found the same mutation characteristic of MEN 2A in both families. We also screened probands from three pedigrees with familial cutaneous lichen amyloidosis for RET mutations. In none of the RET coding and flanking intronic sequences was a mutation detected. This most probably indicates that skin amyloidosis found in some MEN 2A families and familial cutaneous lichen amyloidosis are different conditions. Consequently, patients with apparent familial cutaneous lichen amyloidosis do not appear to be at risk for MEN 2A.

Herpes zoster-varicella in cutaneous T-cell lymphomas.

The overall frequency of herpes zoster-varicella (HZV) infection in 221 patients with histologically confirmed cutaneous T-cell lymphoma was 10% (22 patients). The frequency of HZV infection and serious complications (viral dissemination, bacteremia) was relatively high in patients with evidence of extracutaneous involvement, especially in patients with Sézary syndrome. The major factors identified to account for this predisposition to HZV infection include intensive treatment with radiation therapy or drugs given for systemic effect and/or immunologic deficiency consequent to advanced disease. These observations are quite similar to those made in patients with Hodgkin's disease.

Primary cutaneous large B-cell lymphomas of the legs. A distinct type of cutaneous B-cell lymphoma with an intermediate prognosis. Dutch Cutaneous Lymphoma Working Group.

BACKGROUND AND DESIGN: Primary cutaneous follicular center cell lymphomas represent a distinct type of cutaneous B-cell lymphoma, clinically characterized by localized skin lesions on the head or trunk and an excellent prognosis. Histologically similar lymphomas may occur on the legs. The clinical behavior of this group is still undefined, and controversy exists whether these lymphomas should be classified as follicular center cell lymphoma or B-immunoblastic lymphoma. We reviewed the clinical, histologic, and follow-up data of 18 patients with primary cutaneous large B-cell lymphoma of the legs. RESULTS: Primary cutaneous large B-cell lymphoma of the legs generally occurred in elderly patients (median age at diagnosis, 76 years), in particular women (male-female ratio, 7:2), and preferentially affected the lower legs (14 of 18 patients). Radiotherapy and/or systemic polychemotherapy resulted in complete remissions in 16 of 17 patients. Follow-up data demonstrated estimated 2- and 5-year survival rates of 77% and 58%, respectively. Histologic evaluation showed diffuse dermal infiltrates with variable proportions of centroblasts (large noncleaved cells), large centrocytes (large cleaved cells), and B immunoblasts. Seventeen of 18 patients were diagnosed as having primary cutaneous follicular center cell lymphoma; only 1 patient, whose histologic examination showed more than 30% immunoblasts, was diagnosed as having B-immunoblastic lymphoma. CONCLUSIONS: Primary cutaneous large B-cell lymphoma of the legs is a distinct clinicopathologic entity that mainly affects elderly patients and has an intermediate prognosis. Although most cases have a follicular center cell origin, primary cutaneous large B-cell lymphoma is proposed as the most appropriate term for this type of cutaneous lymphoma.

Mycosis fungoides: disease evolution and prognosis of 309 Dutch patients.

OBJECTIVES: To determine the disease course of Dutch patients with mycosis fungoides and to define factors related to disease progression and survival. DESIGN: A multicenter, 13-year, retrospective cohort analysis. SETTING: Eight dermatology departments collaborating in the Dutch Cutaneous Lymphoma Group. PATIENTS: Three hundred nine patients with mycosis fungoides registered between October 1985 and May 1997, including 89 patients with limited patches or plaques (stage Ia), 135 with generalized patches or plaques (stage Ib), 46 with skin tumors (stage Ic), 18 with enlarged but uninvolved lymph nodes (stage II), 18 with lymph node involvement (stage III), and 3 with visceral involvement (stage IV). MAIN OUTCOME MEASURES: Response to initial treatment, sustained complete remission, actuarial disease progression, and overall and disease-specific survival per clinical stage. RESULTS: The median follow-up was 62 months (range, 1-113 months). For the entire group, the actuarial overall and disease-specific survival was 80% and 89% at 5 years, and 57% and 75% at 10 years, respectively. The actuarial 5-year disease-specific survival of patients with stage Ia, Ib, and Ic disease was 100%, 96%, and 80%, respectively, and only 40% for patients with stage III disease. Using multivariate analysis, the presence of extracutaneous disease, the type and extent of skin involvement, the response to initial treatment, and the presence of follicular mucinosis were independently associated with higher disease progression and mortality rates. The calculated risks of disease progression at 5 and 10 years gradually increased from 4% to 10% for those with stage Ia disease, from 21% to 39% for those with stage Ib disease, and from 32% to 60% for those with stage Ic disease; for those with stage III disease, the risk remained at 70% at 5 and 10 years. The overall risk of disease progression at 5 and 10 years was 24% and 38%, respectively, for the total study group. CONCLUSION: At least within the first 10 years after diagnosis, disease progression and mycosis fungoides-related mortality occur in only a subset of patients generally presenting with advanced disease.

Epidermodysplasia verruciformis: Langerhans cells, immunologic effect of retinoid treatment and cytogenetics.

A case study is presented of a 44-year-old negroid male with epidermodysplasia verruciformis (EV), cutaneous carcinomas, and impaired cell-mediated immunity (CMI), infected with human papillomavirus type 8 and 17. Analysis was made of (a) T6+ and HLA-DR+ Langerhans cells (LCs) by immunoperoxidase staining in lesional and clinically normal skin before and during retinoid treatment, (b) the effect of retinoid treatment on CMI in vivo and in vitro, and (c) cytogenetic aspects related to chromosomal instability. The results showed the virtual absence of T6+ and HLA-DR+ LCs in koilocytic areas of epidermis involved with EV. Light-exposed, clinically normal skin also demonstrated microscopic EV lesions largely devoid of T6+ and HLA-DR+ LCs. Retinoid treatment with etretinate (Ro 10-9359) appeared both to increase the CMI response in vitro to T-cell mitogens and to influence the in situ pattern of T6+ and HLA-DR+ LCs. The cytogenetic study did not show evidence of spontaneous or UV-induced chromosomal instability.

Syphilis management and treatment.

Syphilis poses a serious health problem in many developing countries and in some areas of North America and Europe, especially Eastern Europe. This article initially addresses the state of the art regarding the interaction between syphilis and HIV infection and its consequences for management and treatment. Further attention is given to laboratory diagnosis of syphilis and false-positive and false-negative serologic reactions. The diagnosis and management of neurosyphilis, ocular, cardiovascular, and congenital syphilis are addressed, as well as management of syphilis patients allergic to penicillin and the Jarisch-Herxheimer reaction. Finally, the role of partner(s) and contact tracing is discussed.