Permanent Cures Research Articles

RNA Therapeutics for the Cardiovascular System.

RNA therapeutics hold significant promise in the treatment of cardiovascular diseases. RNAs are biologically diverse and functionally specific and can be used for gain- or loss-of-function purposes. The effectiveness of mRNA-based vaccines in the recent COVID-19 pandemic has undoubtedly proven the benefits of an RNA-based approach. RNA-based therapies are becoming more common as a treatment modality for cardiovascular disease. This is most evident in hypertension where several small interfering RNA-based drugs have proven to be effective in managing high blood pressure in several clinical trials. As befits a rapidly burgeoning field, there is significant interest in other classes of RNA. Revascularization of the infarcted heart through an mRNA drug is under clinical investigation. mRNA technology may provide the platform for the expression of paracrine factors for myocardial protection and regeneration. Emergent technologies on the basis of microRNAs and gene editing are tackling complex diseases in a novel fashion. RNA-based gene editing offers hope of permanent cures for monogenic cardiovascular diseases, and long-term control of complex diseases such as essential hypertension, as well. Likewise, microRNAs are proving effective in regenerating cardiac muscle. The aim of this review is to provide an overview of the current landscape of RNA-based therapies for the treatment of cardiovascular disease. The review describes the large number of RNA molecules that exist with a discussion of the clinical development of each RNA type. In addition, the review also presents a number of avenues for future development.

Antiviral potential of Medicinal plants against Influenza Viruses: A Systematic Review

Influenza is a considerable public health hazard that affects people worldwide. Effective diagnosis and timely treatment are critical for the influenza virus. The current review primarily focuses on isolating a novel, cost-effective anti-influenza viral. Several natural bioactive compounds have precious therapeutic potential. We have shortlisted twenty-one medicinal plants with potent antiviral properties effective at different stages of replication of the influenza virus. Certain extracts of these medicinal plants are gaining popularity compared to synthetic drugs due to their safety and broad-spectrum action. Our meta-analysis focuses on various characteristics of the medicinal plant extracts that help prevent and spread the influenza virus in the early stages. Oseltamivir, zanamivir, amantadine, and rimantadine are used to inhibit the activity of neuraminidase and matrix-2 proteins. However, they have side effects. Their inappropriate doses may cause mutation and confer resistance to the antivirals. According to the circulating strain, the influenza vaccine needs to be updated every year. As a result, chemically synthetic drugs and the current vaccination are not long-term solutions. Thus, we focus on plant metabolites, which are less harmful, permanent cures, and cost-effective than synthetic drugs.

Regulated Expression of GATA1 As a Gene Therapy Cure for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure disorder characterized by impaired red blood cell production leading to severe anemia. The only treatment options are regular transfusions, corticosteroids, or bone marrow transplant, and each has variable effectiveness and considerable morbidity. More effective, safer, and permanent cures for DBA are lacking, but few drugs are in active development. DBA is genetically heterogenous, characterized by loss-of-function mutations in one copy of at least 26 different ribosomal protein (RP) genes that alters the mRNA translation of a select group of genes, most notably the hematopoietic master regulator GATA1. Overexpression of GATA1 in hematopoietic stem and progenitor cells (HSPCs) from patients with DBA rescues the erythroid differentiation defects in vitro, but unregulated GATA1 expression in hematopoietic stem cells (HSCs) promotes premature erythroid differentiation at the expense of long-term (LT-) HSC maintenance. Gene therapy is an attractive strategy to achieve a cure for DBA, but a traditional gene therapy approach that overexpresses a functional copy of a mutated gene is not the ideal option, as it would require the development and validation of dozens of gene therapy vectors, each with a copy of one of the mutated DBA genes. Here, we report a unified gene therapy strategy, using developmentally-regulated and highly restricted expression of GATA1 that would be curative in most, if not all, patients with DBA, regardless of the underlying disease-causing mutation. To be a cure for DBA, a GATA1 gene therapy vector must incorporate into the genome of undifferentiated LT-HSCs but only drive robust GATA1 expression in early erythroid progenitors after lineage commitment. To design a vector with lineage-restricted expression, we identified accessible chromatin regions upstream of GATA1 that only gain accessibility in differentiating erythroid cells, but not in upstream HSPCs. Using these selectively accessible regions concatenated together, we constructed a human GATA1 enhancer (hG1E) element and used it to drive GATA1 and/or GFP expression from a lentiviral backbone. To ensure erythroid-restricted expression of the hG1E-GATA1-GFP vector, we infected CD34+ HSCs from healthy human donors and cultured them in conditions that either allowed HSC maintenance or promoted erythrocyte differentiation. We observed very low GFP expression in bulk HSCs and essentially no expression in the subpopulation of immunophenotypic LT-HSCs. However, more than 80% of committed erythroid cells demonstrated high levels of GFP expression, confirming the fidelity of lineage-restricted expression from the hG1E-GATA1 vector. To examine whether lineage-restricted GATA1 expression confers a functional correction of the erythroid maturation block in DBA, we used CRISPR editing to recapitulate RPS19 haploinsufficiency in human HSPCs. In RPS19 edited samples that were infected with a control GFP virus, we observed a progressive depletion over time of RPS19 edits as the cells underwent erythroid differentiation, consistent with the notion that RPS19 editing leads to impaired erythroid differentiation and are selected against. In contrast, RPS19-edited and hG1E-GATA1 treated samples had a more than 10-fold higher preservation of RPS19 edits in the bulk population and in erythroid colonies derived from single progenitor clones, indicating that regulated GATA1 expression could overcome RPS19 haploinsufficiency. To validate the results from the CRISPR model of DBA, we sought to correct the erythroid maturation block in primary HSCs from a DBA patient with a pathogenic mutation in RPL5. There was skewed differentiation of GFP-infected RPL5+/- HSCs away from the erythroid lineage and toward myeloid and megakaryocyte lineages, but regulated GATA1 expression from the hG1E-GATA1 vector reversed that skewing. There was a 10-fold increase in the ratio of erythroid to non-erythroid cells, and a significant expansion of erythroid cells in the hG1E-GATA1 treated patient cells. Preclinical assessment in additional patient samples both in vitro and in vivo is ongoing. Collectively, our preclinical data demonstrate that lineage-restricted expression of GATA1 is sufficient to rescue the impaired erythroid differentiation in DBA regardless of the underlying genetic lesion. These results will set the stage for the first in-human gene therapy trial for DBA.

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV.

Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

Charmadala: Virudhaahar Janya Kushtha - A Case Study

In the midst of movement and chaos of life, we forget to pay heed to our health and as a result of which susceptibility to various allergens is increasing day by day. Allergens can be dust, smoke, fumes, pollen and even food. A person can either be allergic to certain allergen right from his birth or can acquire hypersensitivity in later stage of his life. Food allergy is quite common in present era and is affecting the lives of many people. Although there are a number of manifestations of food allergy like nausea, vomiting, diarrhoea, headache etc, most commonly seen are skin manifestations like blisters, itching, redness, burning sensation, oozing, discolouration etc. Acharyas have also mentioned Virudhaahar as one of the most prominent cause of Kushtha. Virudhaahar is any food that causes sensitivity reactions in a person. In contemporary sciences, there are no such permanent cures for hypersensitivity induced skin diseases. But in Ayurveda, through Shodhana and Shamana, these can be treated effectively and occurrence can also be avoided to some extent. This case report is of 38 yrs old male who visited our OPD with itching, oozing, blisters with intense burning sensation in palms and redness and itching in groin areas. The disease was diagnosed here as Charmdala. The treatment protocol opted was Virechan and Shamana. The patient was assessed every 7 days for a month and showed significant relief. This case thus proves that Ayurveda has the potential for curing Virudhaahar janya Kushtha which is often unresponsive to the contemporary treatment protocol.

Protective Effects of Korean Herbal Remedy against Airway Inflammation in an Allergic Asthma by Suppressing Eosinophil Recruitment and Infiltration in Lung.

The increasing prevalence of allergic asthma has become the world’s major health issue. Current treatments for allergic asthma focus on treating symptoms, while permanent cures still remain undiscovered. In this study, we investigated the effect of Korean traditional herbal remedy, Pyunkang-tang (PGT)—composed of six plants—on asthma alleviation in a mouse model. The PGT mixture was orally gavaged to mice (PM group, 20 mg/mouse/day) from 7 days before sensitization with ovalbumin (OVA) (day −7). On day 0 and day 14, mice from OVA-control (n = 9) and PM group (n = 8) were sensitized with OVA and alum through intraperitoneal injection. On days 18~20, OVA was challenged to mice through nasal injection and sacrificed next day. Cell profile in lung tissue was analyzed by flow cytometry and RT-qPCR analysis, and the number of eosinophils and expression of siglec-F were significantly reduced in the PM group. Lung tissue was examined with hematoxylin and eosin (H&E) and Alcian blue/periodic acid–Schiff (AB-PAS) staining. Noticeably reduced eosinophil infiltration around bronchioles was displayed in the PM group compared to the OVA-control group. Furthermore, PGT-treated mice showed a significant reduction in IL-13 and a mild reduction in IL-5 in lungs. A decreasing tendency of IL-5/13 (+) CD4+ T cells and IL-13(+) innate lymphoid cells (ILCs) and a significant reduction in IL5(+) ILCs were also observed. When treating PGT on murine lung epithelial cells stimulated by papain, there was a significant reduction in IL-33 mRNA expression levels. Taken together, oral delivery of PGT successfully alleviated asthmatic responses provoked by OVA in a mouse model and could lead to novel therapies for allergic asthma.

Cell-Based Therapy Manufacturing in Stirred Suspension Bioreactor: Thoughts for cGMP Compliance.

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson’s disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the “cells are the final products” and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.

Recent progress in understanding and manipulating haemoglobin switching for the haemoglobinopathies

The major β-haemoglobinopathies, sickle cell disease and β-thalassaemia, represent the most common monogenic disorders worldwide and a steadily increasing global disease burden. Allogeneic haematopoietic stem cell transplantation, the only curative therapy, is only applied to a small minority of patients. Common clinical management strategies act mainly downstream of the root causes of disease. The observation that elevated fetal haemoglobin expression ameliorates these disorders has motivated longstanding investigations into the mechanisms of haemoglobin switching. Landmark studies over the last decade have led to the identification of two potent transcriptional repressors of γ-globin, BCL11A and ZBTB7A. These regulators act with additional trans-acting epigenetic repressive complexes, lineage-defining factors and developmental programs to silence fetal haemoglobin by working on cis-acting sequences at the globin gene loci. Rapidly advancing genetic technology is enabling researchers to probe deeply the interplay between the molecular players required for γ-globin (HBG1/HBG2) silencing. Gene therapies may enable permanent cures with autologous modified haematopoietic stem cells that generate persistent fetal haemoglobin expression. Ultimately rational small molecule pharmacotherapies to reactivate HbF could extend benefits widely to patients.

Abstract 4079: Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers

Abstract Selective High Affinity Ligands (SHALs) are small molecules that can be designed to bind selectively and with high affinity to any protein target by linking together several ligands that recognize and bind to different sites on the protein’s surface. SH7139, our first SHAL therapeutic for treating advanced non-Hodgkin’s lymphoma, has shown remarkable efficacy in B-cell lymphoma xenograft models by providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.4 μg/kg. Functioning similar to an antibody-drug conjugate and a pro-drug, SH7139 targets and binds to a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope are expressed in approximately 85% of B-cell lymphomas. Upon binding to its HLA-DR target, SH7139 is carried into the cytoplasm where it is concentrated and subsequently metabolized, releasing toxic metabolites (derived from each of the linked ligands) that inhibit a series of cellular activities required for tumor cell function. In preparation for an IND application submission and advancing SH7139 into clinical trials, acute dose range finding (i.v. bolus) and multiple dose (i.v. bolus on days 1, 8, and 15) toxicology and safety studies have been conducted with SH7139 in rats and beagle dogs. In the rat studies, doses up to 30 mg/kg (NOAEL = 30 mg/kg, 11,854 fold higher than the efficacy dose), were well tolerated with no findings associated with the drug. Beagle dogs were found to be the most sensitive species (NOAEL = 0.3mg/kg, 395 fold higher than the efficacy dose), with doses at 1 and 10mg/kg producing a reversible allergic-type reaction. A maximum tolerated dose (MTD) was reached in rats (100 mg/kg). No attempt was made to identify the MDT in dogs. In vitro assays conducted to evaluate potential cardiac safety showed SH7139 had no effect on potassium (hERG) and calcium (hCaV1.2) ion channel function up to the highest concentration tested (25 μM). A detectable effect above background was only observed with the sodium channel hNaV1.5 at 25 μM, a plasma concentration that would only be achieved in patients administered a dose ~2,000 fold higher than the efficacy dose. SH7139 was found to be negative in inducing gene mutation in five tester strains of Salmonella and E. coli (AMES assay). In a CHO-K1 micronucleus assay, no genotoxicity was observed in the presence of S9 over the entire concentration range tested. In the absence of S9, a positive response (3.3 fold) just above the triggering threshold (3 fold) of the assay was observed at the highest SH7139 concentration (500 μM). Based on these results and others derived from mouse xenograft efficacy studies, a maximum recommended Phase I trial starting dose has been tentatively set at 0.5 μg/kg with a safety factor of 324. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Rod Balhorn, Monique Cosman Balhorn. Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2017-4079

Abstract 1171: Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors

Abstract SH7139, the first of a new class of cancer therapeutics developed for treating non-Hodgkin’s lymphoma, is unusual in that both targeting and pro-drug functionalities have been incorporated into the same small molecule. Functioning similar to an antibody-drug conjugate, SH7139 targets a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope within the β-subunit are reported to be over-expressed in approximately 85% of B-cell lymphomas. Upon binding to HLA-DR molecules located on the tumor cell’s surface, SH7139 is transported into the cytoplasm where it is concentrated and subsequently metabolized. A series of metabolic products derived from the SHAL’s recognition elements (three small molecules that are linked together to create SH7139 and to provide targeting selectivity) are generated as the drug is broken down, each of which inhibits one or more activities required for tumor cell survival. Preclinical studies with SH739 have demonstrated remarkable efficacy in treating B-cell lymphoma xenografts in mice, providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.41 μg/kg. Biopsy tissue binding studies conducted with SH7129, a biotinylated form of SH7139, and streptavidin-horse radish peroxidase detection have shown the drug binds to a significant fraction of tumors obtained from patients diagnosed with multiple myeloma and each of the B-cell lymphoma subtypes tested to date (DLBC, Burkitt’s, Mantle Cell, Follicular, MALT, and CLL). SH7129 was also observed to bind to tumor biopsies obtained from of a number of patients diagnosed with peripheral T-cell and nodular sclerosis Hodgkin’s lymphomas, a result consistent with observations reported by others that HLA-DRs are expressed in a subset of these lymphomas. HLA-DR expression has also been reported to occur in or be linked to a number of other types of cancer, including melanoma, cervical, ovarian, pancreatic and lung cancers. SH7129 staining of tumor microarrays have shown biopsy cores from a subset of patients diagnosed with each of these cancers also bind SH7139. While in vivo efficacy has only been tested in Burkitt’s (Raji), Mantle cell (Granta-519), and T-cell (Jurkat, a cell line control lacking HLA-DR and showing no efficacy) lymphoma xenografts, these tissue binding results suggest that in addition to the majority of the NHL subtypes, nodular sclerosis Hodgkin’s lymphoma, multiple myelomas, as well as a subset of melanomas, ovarian, cervical, pancreatic, and lung cancers may also respond to SH7139 therapy. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Monique Cosman Balhorn, Rod Balhorn. Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2017-1171

Demonstrating Potential of Cell Therapy for Wilson's Disease with the Long-Evans Cinnamon Rat Model.

Wilson's disease (WD) is characterized by the inability to excrete copper (Cu) from the body with progressive tissue injury, especially in liver and brain. The molecular defect in WD concerns mutations in ATP7B gene leading to loss of Cu transport from the hepatocyte to the bile canaliculus. While drugs, e.g., Cu chelators, have been available for several decades, these must be taken lifelong, which can be difficult due to issues of compliance or side effects. Many individuals may require liver transplantation, which can also be difficult due to donor organ shortages. Therefore, achieving permanent cures via cell or gene therapy are of great interest for WD. Cell therapy is feasible because transplanted hepatocytes can integrate in liver parenchyma and restore deficient functions, including transport of Cu into bile. The availability of authentic animal models that recapitulate hepatic WD, especially the Long-Evans Cinnamon (LEC) rat, has advanced cell transplantation research in WD. We describe requirements for cell therapy in animal models with several standardized methods for studies to test or refine cell therapy strategies in WD.

Controversies in IgG replacement therapy in patients with antibody deficiency diseases

This Current perspectives article will review and highlight the importance of accurate diagnosis of patients who have failed to produce specific antibodies to naturally encountered foreign proteins or polysaccharides or after vaccination and the appropriate institution of immunoglobulin replacement therapy. The field of primary immunodeficiency disease (PIDD) has expanded remarkably since the early descriptions 6 decades ago. With greater recognition and advanced cellular and molecular diagnostic technology, new entities and single-gene defects in patients with PIDD are rapidly being defined. This, combined with treatment advances and newborn screening for severe combined immunodeficiency, has resulted in improved outcomes and survival and even permanent cures. Awareness of PIDD has also increased, but the guidelines for recognition remain to be validated. The zeal for registering and enrolling patients has potentially created a large body of "patients" treated with immunoglobulin replacement unnecessarily. The complexity, diversity, and availability of laboratory testing have brought awareness of PIDD to the forefront, but because of an absence of standardization of certain assays, concerns about the correct diagnosis and appropriate treatment have increased. We hope to refocus the discussion on identifying clear laboratory and clinical guidelines for the establishment of an accurate diagnosis of antibody deficiency, its rationale, and, where indicated, institution of safe treatment.

Highly Efficient Zinc-Finger Nuclease-Mediated Disruption of an eGFP Transgene in Keratinocyte Stem Cells without Impairment of Stem Cell Properties

Zinc-finger nucleases (ZFNs) are sequence-specific genome engineering tools with great potential for the development of gene therapies. The achievement of permanent cures through gene therapy requires targeting of stem cells but the effects and/or side effects of ZFN treatment on adult stem cell potency are largely unknown. Keratinocyte stem cells (KSCs) are attractive candidates for the development of gene therapies as their isolation, culture and grafting are well established. We derived KSCs from eGFP-transgenic mice and knocked out eGFP expression by disrupting the open reading frame with specific ZFNs in cell culture. EGFP-negative KSCs were then used as a model system to study the impact of ZFN treatment on stem cell potential. We achieved high gene disruption efficiencies with up to 18% eGFP-negative KSCs. As expected, ZFN cytotoxicity increased with rising ZFN concentrations. However, the ratio of correctly targeted KSCs among total treated cells was similar at different ZFN doses. Most importantly, our in vitro assays showed that ZFN-treated KSCs maintained their stem cell potential. They retained the capacity to both self-renew and form fully differentiated epidermal equivalents in culture. Moreover, they were able to form spherical aggregates in suspension culture, a characteristic hallmark shared with other stem cell types, and they expressed the in vivo KSC markers K15, NFATc1 and Sox9. Our data suggest that the stem cell potential of KSCs is not impaired by highly efficient ZFN treatment.

Boris Bastian

Boris Bastian

A Spiritual Healing Mission Remembered: James Moore Hickson's Christian Healing Mission at Palmerston North, New Zealand, 1923

The 1923 world tour by the Anglican spiritual healer James Moore Hickson finished in New Zealand. Coming at a time of heightened interest in the phenomenon of spiritual healing, Hickson attracted large crowds and intense media attention. Because so many of those who came for healing came from outside the city, the mission at All Saints' Anglican Church in the provincial city of Palmerston North involved a disproportionate effort on the part of parishioners compared with other centres. So, although the number of permanent cures was not as high as was first hoped, and the mission did not lead to increased participation in church activities, the event became a significant part of the history of All Saints'. As elsewhere, some individuals were deeply affected, many claiming to experience a new depth in their spiritual lives. These individuals, and stories of the Hickson mission, encouraged openness to spiritual healing and charismatic renewal that continues to influence this particular church to the present day.

Photodynamic therapy plus low-dose cyclophosphamide generates antitumor immunity in a mouse model

Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of nontoxic photosensitizers with harmless visible light-producing cytotoxic reactive oxygen species. PDT causes apoptosis and necrosis of tumor cells, destruction of the tumor blood supply, and activation of the immune system. The objective of this study was to compare in an animal model of metastatic cancer PDT alone and PDT combined with low-dose cyclophosphamide (CY) a treatment that has been proposed to deplete regulatory T cells (T-regs) and increase the immune response to some tumors. We used J774 tumors (a highly metastatic reticulum cell sarcoma line) and PDT with benzoporphyrin derivative monoacid ring A, verteporfin for injection (BPD; 1-mg/kg injected i.v. followed after 15 min by 150 J/cm(2) of 690-nm light). CY (50 or 150 mg/kg i.p.) was injected 48 h before light delivery. PDT alone led to tumor regressions and a survival advantage but no permanent cures were obtained. BPD-PDT in combination with low-dose CY (but not high-dose CY) led to 70% permanent cures. Low-dose CY alone gave no permanent cures but did provide a survival advantage and was shown to reduce CD4+FoxP3+ T-regs in lymph nodes, whereas high-dose CY reduced other lymphocyte classes as well. Cured animals were rechallenged with J774 cells, and the tumors were rejected in 71% of mice. Cured mice had tumor-specific T cells in spleens as determined by a (51)Cr release assay. We conclude that low-dose CY depletes T-regs and potentiates BPD-PDT, leading to tumor cures and memory immunity.

Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors.

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.

Directed gene modification via triple helix formation.

The ability to selectively target mammalian genes and disrupt or restore their function would represent an important advance in gene therapy. Mutation of a single nucleotide can often result in a non-functional gene product. Reversion of defective genes to their correct sequences could lead to permanent cures for patients with many genetic diseases. Molecules such as triplex forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) are currently being employed to bind to double-stranded DNA. Efficient targeting of genomic DNA with these molecules will be the initial step in gene modification.

Introduction: Changing Trends in Antiarrhythmic Therapy: Worldwide Experience With Class III Agents

T approaches to cardiac arrhythmias continue to change rapidly, as indicated by the recent acceptance of a number of revolutionary concepts. These include (1) the recognition that time-honored antiarrhythmic drugs that act essentially by slowing cardiac conduction (class I agents) may increase mortality despite suppressing cardiac arrhythmias. The result has been a shift to agents that act largely by prolonging cardiac repolarization (class III agents); (2) the realization that radiofrequency catheter ablation may produce permanent cures in many forms of arrhythmia, especially those of supraventricular origin; and (3) the introduction and refinement of the implantable cardioverter-defibrillator (ICD), which has the potential for prolonging survival by terminating ventricular tachycardia and fibrillation (VT/VF). The changing trends in the use of pharmacologic agents and advances in catheter ablation and ICD technology are profoundly impacting how physicians are deciding to terminate and/or prevent specific types of arrhythmia, not only to reduce or eliminate symptoms but also to prolong survival by reducing the probability of arrhythmic death. It has, however, become increasingly clear that survival in patients with arrhythmias cannot always be measured accurately by quantifying presumed arrhythmic deaths. The outcome that best indicates improved survival on antiarrhythmic therapy is the decrease in all-cause or total mortality, now the decisive endpoint of all mortality trials, whether they involve ICDs or antiarrhythmic drugs. For the most part, progress in antiarrhythmic drug therapy has been driven by data generated by randomized, controlled From the Division of Cardiology and the Department of Medicine, Veterans Affairs Medical Center of West Los Angeles, and the Department of Medicine, University of California, Los Angeles School of Medicine, Los Angeles, California. Address for reprints: Bramah N. Singh, MD, PhD, Division of Cardiology (111E), Veterans Affairs Medical Center of West Los Angeles, 11301 Wilshire Boulevard, Los Angeles, California 90073.

The future of community care.

BrMedJ 1990;300:923-4 Community care is not working. The government has decided to stall its programme of hospital closures (box), and the virtues of asylum care are being extolled once more.1 In the jargon of planners, reprovision is not keeping pace with deinstitutionalisation. Former patients have ended up on the streets2 and in prisons,1 prompting great public and political concern. But the biggest scandal of community care is that it has not been monitored adequately. Nearly 100000 long patients have been discharged from Britain's mental hospitals in the past 35 years and fewer than 4000 places have been provided in local authority hostels. These statistics, which are much quoted, do not necessarily mean that community care has failed the remaining patients. The numbers simply tell us that the Department of Health does not know what has happened to that invisible cohort. Probably some of these people live with their families, some in hostels run by voluntary or private organisations, and some in independent accommodation. And some must be dead, because they were elderly when discharged. But no one knows for sure. Community care is not needed only for former residents of large mental institutions. Young people with relapsing psychotic illnesses and related long term disabilities?the long stay population4? still need shelter and rehabilitation. Champions of community care have been too optimistic, hoping that neuroleptic drugs and new psychological treatments might provide permanent cures for all.