Ancient medicine for a modern disease: traditional Amazonian medicine to treat substance use disorders.

Background: There exists an underexploited opportunity to develop innovative therapeutic approaches to SUDs based upon the complementarity between modern and traditional health systems.Objectives: Illustrate the feasibility and potentiality of such an approach through the comprehensive description of Takiwasi Center's treatment model and program, where health concepts and practices from traditional Amazonian medicine work synergistically with modern psychotherapy and medicine in an intercultural dialog to assist in the rehabilitation of people suffering from SUDs.Methods: The description was built from a review of the literature, institutional data, participatory observation and unstructured interviews with staff, researchers and patients during treatment.Results: Since the foundation of the Takiwasi Center in 1992 in the peruvian Amazon, more than a thousand patients with different socio-cultural, ethnic and religious backgrounds have received residential treatment. We present how traditional Amazonian medicine techniques and health concepts cooperate to complement modern psychology in a therapeutic community setting and propose some hypotheses about the neurobiological, psycho-emotional and spiritual healing mechanisms triggered by the program to help people identify and heal the roots of their substance misuse and addictive behavior. We also summarize quantitative outcomes during treatment showing significant improvements in a wide variety of mental health indicators.Conclusion: Takiwasi Center's program is an option for people seeking non-conventional treatment who are sensitive to traditional Amazonian medicine practices and ready to explore the roots of their addiction. From this intercultural approach, some lessons could emerge toward a broader understanding of SUDs that may result in better patient care.

Ayahuasca Treatment Outcome Project (ATOP): One Year Results from Takiwasi Center and Implications for Psychedelic Science

OBJECTIVE: This paper focuses on the outcomes at one-year post-treatment of a naturalistic evaluation of services provided through the Takiwasi Centre, an accredited Peruvian therapeutic community offering an ayahuasca-assisted, integrative treatment program for addiction rehabilitation. METHODS: Participants (n=52) completed structured interviews and a battery of validated instruments. Outcome measures included the Addiction Severity Index (V. 5), the Beck Anxiety and Depression Inventories, the WHOQOL-BREF and the WHOQOL-SRPB. Likert rating scales were used to assess perceived importance and significance of different aspects of the program and overall participant satisfaction. RESULTS: The group change from baseline to the one-year follow up was significant and in the anticipated direction for alcohol and drug use severity, depression and anxiety, and some dimensions of quality of life. There was considerable individual variation in outcomes and treatment duration. The majority of participants rated all aspects of the program as important, including the spiritual and therapeutic significance of the ayahuasca experience which was rated as very significant. CONCLUSION: Based on the positive one-year outcomes, and within the limitations of an uncontrolled observational study design, the findings suggest promise for the effectiveness of the use of ayahuasca in a multifactorial treatment context for individuals with significant treatment histories, high levels of comorbidity and treatment motivation. Results highlight considerable variation in individual experience that merit in-depth qualitative analysis. Implications for ayahuasca-assisted and other psychedelic-assisted treatment alternatives are discussed.

Protocol for Outcome Evaluation of Ayahuasca-Assisted Addiction Treatment: The Case of Takiwasi Center.

The present study describes the protocol for the Ayahuasca Treatment Outcome Project (ATOP) with a special focus on the evaluation of addiction treatment services provided through Takiwasi Center, the first ATOP study site. The goal of the project is to assess treatment outcomes and understand the therapeutic mechanisms of an Ayahuasca-assisted, integrative treatment model for addiction rehabilitation in the Peruvian Amazon. The proposed intervention protocol highlights the significance of treatment setting in the design, delivery, and efficacy of an addiction rehabilitation program that involves the potent psychedelic tea known as Ayahuasca. After describing the context of the study, we put forth details about our mixed-methods approach to data collection and analysis, with which we seek to gain an understanding of why, how, and for whom this specific ayahuasca-assisted treatment program is effective across a range of outcomes. The ATOP protocol employs qualitative research methods as a means to determine which aspects of the setting are meaningful to clients and practitioners, and how this may correlate with outcome measures. This paper delineates the core principles, methods, and measures of the overall ATOP umbrella, then discusses the role of ATOP in the context of the literature on long-term residential programs. To conclude, we discuss the strengths and limitations of the protocol and the intended future of the project.

Pilot Evaluation of a Residential Drug Addiction Treatment Combining Traditional Amazonian Medicine, Ayahuasca and Psychotherapy on Depression and Anxiety.

Recent research highlighted the therapeutic potential of ayahuasca, a psychoactive plant brew used ritually in traditional Amazonian medicine (TAM). The present study evaluates the impact of integrating ayahuasca and TAM with psychotherapy on depression and anxiety in an inpatient addiction treatment program. Male patients (N = 31) were evaluated pre and post treatment using the Beck Anxiety Inventory (BAI) and the Beck Depression Inventory (BDI). Clinical and sociodemographic characteristics, motivation, quality of life, spirituality, and treatment satisfaction were also measured and analyzed by means of two tailed t-test, one way ANOVA and Spearman test. From pre- to post-treatment, patients showed significant reductions in scores of anxiety (from 20.8 to 11.6, p < .002) and depression (from 18.7 to 7.5, p <.001). Similarly, patients showed higher scores of quality of life (p < .001) and spirituality (p < .001) upon discharge, which correlated with their reduction in scores of anxiety and depression. While future results will evaluate the efficacy of this treatment on measures of addiction at follow-up, the present results build upon previous research to bring further support to the use of Ayahuasca and Amazonian medicine in mental health treatments with a transpersonal focus.

Implementation of Nagoya Protocol on access and benefit-sharing in Peru: Implications for researchers.

ETHNOPHARMACOLOGICAL RELEVANCE: The Peruvian Amazon holds more than 1000 plant species with commercial potential and the national sales of natural products derived from medicinal and aromatic plants have exceeded $ 400 million per year. Research and development activities carried out on the genetic and biochemical composition of Peruvian flora have to abide by national and international regulations, such as the Nagoya Protocol (NP). AIM OF THE STUDY: The aim of this paper is to describe the implications of the current implementation of the NP in Peru for performing research on national genetic resources. MATERIALS AND METHODS: A review of the current legal framework and status of the NP in Peru was performed accompanied by first-hand experience undertaken by submitting a request for access to genetic resources related to wild continental species. RESULTS: So far, Peru has issued 16 Internationally Recognized Certificates of Compliance (IRCCs) through 2 of the identified National Authorities. Some of the difficulties and challenges observed have to do with the degree of effective implementation of the Access and Benefit-Sharing (ABS) system, the fact that the application process is not sufficiently clear, and the wide gap between this formal system and what occurs informally outside of it. In response to this, training and implementation projects have been launched and a new law on the access to genetic resources has been proposed. CONCLUSIONS: The difficulties observed still represent an obstacle to scientific research and the development of new commercial products based on Peruvian traditional knowledge and genetic resources. Although improvements have been made to the ABS framework, there remain major challenges to encouraging and ascertaining the equitable and sustainable use of Peru's biodiversity.

Short-Term Treatment Effects of a Substance Use Disorder Therapy Involving Traditional Amazonian Medicine.

Chronic illness management today commonly involves alternative medicines. Substance use disorder (SUD), as a chronic psychosomatic illness, might benefit from a similar approach. The accredited Takiwasi Center offers such an SUD treatment program involving Amazonian medicine combined with psychotherapy. The current study assessed this integrative program's short-term therapeutic effects. We measured baseline data from 53 dependence-diagnosed males admitted to treatment (T1) and repeated clinical outcome variables at treatment completion (T2). Paired samples t-tests were used to assess changes between T1 and T2 (n = 36). Nearly all participants (age M= 30.86, SD= 8.17) were dependent on multiple substances, most prominently cannabis, alcohol, and cocaine-related drugs. A significant decrease (T1 to T2) was found for addiction severity outcomes drug use (p < .001), alcohol use (p < .001), psychiatric status (p < .001), and social/familial relationships (p < .001). Emotional distress also diminished significantly (p < .001), as did substance craving (p < .001). Quality of life increased significantly from T1 to T2 (p < .001). Our results provide first indications for significantly improved SUD symptoms after the Amazonian medicine-based treatment. These findings are preliminary given the design, but strongly encourage further investigation of this therapy, which in the long term may open new therapeutic avenues for SUDs.

Conceptions and practices of an integrative treatment for substance use disorders involving Amazonian medicine: traditional healers' perspectives

Objective: The harmful use of psychoactive substances represents one of today's largest public health problems. Yet, in spite of its global relevance, current treatment for substance use disorders (SUDs) is still not entirely successful. The purpose of this study was to investigate alternative treatments and conceptions from traditional Amazonian medicine adapted to SUDs. Methods: We conducted semi-structured interviews with 13 practicing experts at a well-established addiction treatment center in the Peruvian Amazon and performed qualitative content analysis on the collected data. Main categories were deductively defined and corresponding subcategories inductively developed. Results: Our findings revealed characteristic features and consequences, causes and antecedents, and treatment methods of SUDs as the main categories. Overall, concepts of disease etiology bore resemblance with contemporary biopsychosocial models of SUDs. The Amazonian therapeutic means however differed markedly from current Western ones. The main methods involved dietary retreats, healing ceremonies, and purging rituals. The integral application of Amazonian methods, as well as their traditional implementation according to prescribed ritual protocols, were emphasized by the experts as crucial for efficacy and safety of treatment delivery. Conclusion: We suggest further scientific attention to these therapies, including clinical studies, for which our results provide conceptual underpinnings. Findings from this research expand the cross-cultural understanding of SUDs and, in the long run, may enhance its treatment options.

Conceptions and practices of an integrative treatment for substance use disorders involving Amazonian medicine: traditional healers' perspectives.

OBJECTIVE: The harmful use of psychoactive substances represents one of today's largest public health problems. Yet, in spite of its global relevance, current treatment for substance use disorders (SUDs) is still not entirely successful. The purpose of this study was to investigate alternative treatments and conceptions from traditional Amazonian medicine adapted to SUDs. METHODS: We conducted semi-structured interviews with 13 practicing experts at a well-established addiction treatment center in the Peruvian Amazon and performed qualitative content analysis on the collected data. Main categories were deductively defined and corresponding subcategories inductively developed. RESULTS: Our findings revealed characteristic features and consequences, causes and antecedents, and treatment methods of SUDs as the main categories. Overall, concepts of disease etiology bore resemblance with contemporary biopsychosocial models of SUDs. The Amazonian therapeutic means however differed markedly from current Western ones. The main methods involved dietary retreats, healing ceremonies, and purging rituals. The integral application of Amazonian methods, as well as their traditional implementation according to prescribed ritual protocols, were emphasized by the experts as crucial for efficacy and safety of treatment delivery. CONCLUSION: We suggest further scientific attention to these therapies, including clinical studies, for which our results provide conceptual underpinnings. Findings from this research expand the cross-cultural understanding of SUDs and, in the long run, may enhance its treatment options.

Three-dimensional reconstruction of efferent ducts in wild-type and Lgr4 knock-out mice.

We have recently shown that Lgr4 knock-out (LGR4KO) male mice are infertile due to a developmental defect of the reproductive tract. Spermatozoa do not reach the epididymis and accumulate at the rete testis and efferent ducts (ED). We have proposed that in LGR4KO, ED might fail to connect resulting in blind-ended tubes that preclude the normal transit of sperm cells. To explore this possibility, we reconstructed the three-dimensional (3D) structure of the organ from serial microphotographs. The resulting model allowed to individualize and follow each ED from the testis up to the epididymis, and to display the spatial distribution of their content. The transit of spermatozoa is indeed blocked in LGR4KO mice but, contrary to the expectation, the ducts connect normally to each other, forming a single tube that flows into the epididymis, as in the wild-type animals. In the KO however, transit of the sperm is abruptly blocked at the same level syncytial-like aggregates appear in the luminal space. The model also allowed calculating, for the first time, morphometric parameters of the mouse ED, such as total volume, surface, radius, and length. These data unambiguously showed that ED in the mutant mouse are dramatically shortened and less convoluted than in the wild-type animal, providing an explanation to the phenotype observed in LGR4KO. Combined with in situ immunodetection or RNA in situ hybridization, 3D reconstruction of serial histological sections will provide an efficient mean to study expression profiles in organs which do not lend themselves to whole-mount studies.

Evolutionary conservation of bursicon in the animal kingdom.

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.

Defective postnatal development of the male reproductive tract in LGR4 knockout mice.

The final outcome of tube elongation and branching is to maximize the epithelial exchange surfaces in tubular organs. The molecular and cellular basis of these processes is actively studied in model organs such as mammary glands, liver and kidney, but they remain almost unexplored in the male reproductive tract. Here, we report that the orphan G protein-coupled receptor LGR4/GPR48 plays a role in the postnatal tissue remodeling needed for elongation and convolution of the efferent ducts and epididymis. In LGR4 knockout male mice, tube elongation fails, resulting in a hypoplastic and poorly convoluted tract. Cell proliferation is dramatically reduced in KO affected tissues, providing an explanation to the observed phenotype. Detailed analysis showed that LGR4 inactivation manifests differently in the affected organs. In efferent ducts, immune cells infiltrate the epithelium and reach the lumen, blocking the transit of sperm and testicular fluid. In addition, the hypoplasia and low convolution result in a reduction of the epithelial area involved in liquid reabsorption. Both phenomena contribute in tissue swelling upstream the blockade due to liquid and sperm accumulation, with secondary damaging effects on the germinal epithelium. In the epididymis, the thin and highly convoluted duct is replaced by a large cystic tube which is surrounded by a thick condensation of mesenchymal cells. The abnormal organization of the cellular compartments in and around the ducts suggests that LGR4 might play a role in epithelial-mesenchymal interactions. Altogether, our data identify LGR4 as an important signaling molecule implicated in the tube morphogenesis of the male reproductive tract.

Nonsense-associated alternative splicing of the human thyroglobulin gene.

INTRODUCTION: We have described in previous articles a nonsense mutation (4588C>T, R1511X) in exon 22 of the thyroglobulin (TG) gene in a member of a family with a complex history of congenital goiter. In the mutated thyroid gland, full-length thyroglobulin mRNA is almost undetectable. However, a smaller transcript is detected in which the mutated exon 22 is skipped and the reading frame restored. It is conceivable that alternative splicing might be a mechanism involved in the rescue of nonsense mutations. METHODS: To investigate whether the detection of the alternative mRNA is due to an increase in its concentration or its preferential amplification during reverse transcriptase-PCR in the absence of the normal full-length mRNA competitor, we set up an assay in which the competitor mRNA was provided. We also studied the effect of the 4588C>T mutation on exon definition and processing using wild-type and mutated minigenes. RESULTS: The detection of the alternative mRNA lacking exon 22 is not caused by the absence of the full-length competitor. In contrast, our results demonstrate that the alternative transcript preferentially accumulates in the mutated thyroid at a level similar to the full-length transcript in control tissue. Transient expression experiments with wild-type and mutated minigenes indicate that the mutated exon is as efficiently spliced as the wild-type, suggesting that the 4588C>T mutation does not interfere with exon 22 definition and processing. CONCLUSIONS: The alternative splicing of the TG gene described in this article constitutes a new case of nonsense-associated alternative splicing. We have shown that the mutation itself does not interfere with exon definition and processing in vitro. Our results support the hypothesis that the alternative splicing of the mutated exon is driven by the interruption of the reading frame.

Specificity and promiscuity of gonadotropin receptors.

The dichotomy between hormone recognition by the ectodomain and activation of the G protein by the rhodopsin-like serpentine portion is a well established property of glycoprotein hormone receptors. The specificity barrier avoiding promiscuous activation of the FSH receptor by the high concentration of human chorionic gonadotropin (hCG) prevailing during human pregnancy was thus believed to lie in the ectodomain. In the past two years, mutations responsible for rare spontaneous cases of ovarian hyperstimulation syndromes have partially modified this simple view. Five naturally occurring mutations have been identified which cause an increase in the sensitivity of the FSH receptor to hCG. Surprisingly, these mutations are all located in the serpentine portion of the receptor. In addition to their effect on sensitivity to hCG, they increase sensitivity of the FSH receptor to TSH, and are responsible for activating the receptor constitutively. Together, the available information indicates that the ectodomain and the serpentine domain of the FSH receptor each contribute to the specificity barrier preventing its spurious activation by hCG. While the former is responsible for establishment of binding specificity, the latter introduces a novel notion of functional specificity. Recent data demonstrate that LH and FSH receptors can constitute functional homo- and heterodimers. This suggests the possibility that in cells co-expressing the two receptors, such as granulosa cells, the heterodimers might be endowed with functional characteristics different from those of each homodimer.

Expression pattern of the orphan receptor LGR4/GPR48 gene in the mouse.

Leucine-rich G-protein-coupled Receptors (LGR) constitute a subfamily of receptors related to glycoprotein hormone receptors. Amongst them, LGR4, LGR5 and LGR6 form a cluster for which natural agonists are still unknown. By an extensive gene trapping approach, Leighton et al. (2001) obtained a mouse line in which the LGR4 gene is disrupted by a trap vector carrying two biological markers, beta-geo (a fusion between bacterial beta-galactosidase and neomycin phosphotransferase) and a placental alkaline phosphatase (PLAP). Due to perinatal lethality, characterization of adult mice homozygous for this insertion has been impaired. In the present study we have investigated LacZ and PLAP activity patterns in heterozygous mice as a marker for LGR4 natural expression at both macroscopic and histological levels. We present a detailed atlas of LGR4 expression, which displays very wide expression with particularly strong activity in cartilages, kidneys, reproductive tracts and nervous system cells.

Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography.

An engineered horseradish peroxidase isozyme C (HRP C) gene was constructed by the addition of a 6xArg fusion tail to 6xHis-HRP C by the PCR strategy. The 6xHis-6xArg-HRP C cDNA was expressed in the Sf9 insect cell line from Spodoptera frugiperda infected with Autographa californica nuclear polyhedrosis virus. The recombinant peroxidase isoelectric point was 9.5 as judged by isoelectric focusing and was purified directly from the culture medium at day-6 post-infection by cation-exchange chromatography or immobilised metal ion-affinity chromatography. While the former technique gave a yield of 98.5% with a purification factor of 130, the latter gave only a 68% yield with a purification factor of 140. Results obtained provide evidence that the poly-Arg tag is more effective than the poly-His tag for peroxidase purification from a baculovirus expression system.

Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2.

Bursicon is a neurohumoral agent responsible for tanning and hardening of the cuticle and expansion of the wings during the final phase of insect metamorphosis. Although the hormonal activity was described more than 40 years ago, the molecular nature of bursicon has remained elusive. We identify here Drosophila bioactive bursicon as a heterodimer made of two cystine knot polypeptides. This conclusion was reached in part from the unexpected observation that in the genome of the honey bee, the orthologs of the two Drosophila proteins are predicted to be fused in a single open reading frame. The heterodimeric Drosophila protein displays bursicon bioactivity in freshly enclosed neck-ligated flies and is the natural agonist of the orphan G protein-coupled receptor DLGR2.

Identification and characterization of a novel large insertion/deletion polymorphism of 1464 base pair in the human thyroglobulin gene.

We identified a novel large insertion/deletion (Indel) polymorphism of 1464 bp localized in intron 18 of the human thyroglobulin gene. Data from sequence showed a high A+T content (62%), two 17-bp long motif repeats, and three different types of 10-bp long palindromic sequences. The comparison between these 1464 bp and sequences deposited in National Center for Biotechnology Information (NCBI)/GenBank database exhibit a nonsignificant degree of homology with any previously described sequences. The long polymerase chain reaction (PCR) method was used to amplify the genomic DNA region containing intron 17/exon 18/intron 18/exon 19/intron 19 by primers situated in the introns 17 and 19. The amplification generates two fragments of 3.5 and 5.0 kb that correspond to the exclusion or inclusion of a 1464-bp segment, respectively. Both variants are thus widely represented in the human population; giving allele frequencies of 0.56 (insertion) and 0.44 (deletion). Finally, the polymorphism was confirmed by sequence analysis of the 5.0- and 3.5-kb amplified fragments.

Genotyping and characterization of two polymorphic microsatellite markers located within introns 29 and 30 of the human thyroglobulin gene.

The purpose of the present work was to characterize two new polymorphic microsatellite markers in the thyroglobulin gene. TGrI29 and TGrI30 repeats are located within introns 29 and 30, respectively. Genetic studies were carried out by using polymerase chain reaction (PCR) followed by denaturing polyacrilamide gel electrophoresis. TGrI29 exhibited clearly 4 distinguishable alleles ranging from 197 to 203 base pair (bp) in length and TGrI30 showed 8 alleles ranging from 502 to 542 bp. We characterized the two markers by determinating allele frequencies and measures of variation. The heterozygosities (HET) observed of TGrI29 and TGrI30 were 0.859 and 0.522, respectively. The polymorphism information contents (PIC) were 0.471 and 0.434, respectively. No significant differences from Hardy-Weinberg values were found for these two systems. The PCR products of each allele were cloned using the pGEM-T Easy vector and directly sequenced by Taq polymerase-based chain terminator method. Sequencing analysis indicated that both loci are complex repeats, TGrI29 containing two types of variable motifs (tc)n and (tg)n, and TGrI30 a tetra-nucleotide tandem units (atcc)n. In two TGrI29 alleles and one TGrI30 allele were found two different subtypes in each one, with the same molecular weights but different distribution of the tandem repeats. In conclusion, both microsatellites analyzed are highly informative polymorphic markers and can be used in linkage studies in families with congenital hypothyroidism or autoimmunity thyroid diseases.