Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization.

The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585 and 591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584KW585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591PYRKR595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.

Combining Double-Dose and High-Dose Pulsed Dapsone Combination Therapy for Chronic Lyme Disease/Post-Treatment Lyme Disease Syndrome and Co-Infections, Including Bartonella: A Report of 3 Cases and a Literature Review.

Three patients with relapsing and remitting borreliosis, babesiosis, and bartonellosis, despite extended anti-infective therapy, were prescribed double-dose dapsone combination therapy (DDDCT) for 8 weeks, followed by one or several two-week courses of pulsed high-dose dapsone combination therapy (HDDCT). We discuss these patients' cases to illustrate three important variables required for long-term remission. First, diagnosing and treating active co-infections, including Babesia and Bartonella were important. Babesia required rotations of multiple anti-malarial drug combinations and herbal therapies, and Bartonella required one or several 6-day HDDCT pulses to achieve clinical remission. Second, all prior oral, intramuscular (IM), and/or intravenous (IV) antibiotics used for chronic Lyme disease (CLD)/post-treatment Lyme disease syndrome (PTLDS), irrespective of the length of administration, were inferior in efficacy to short-term pulsed biofilm/persister drug combination therapy i.e., dapsone, rifampin, methylene blue, and pyrazinamide, which improved resistant fatigue, pain, headaches, insomnia, and neuropsychiatric symptoms. Lastly, addressing multiple factors on the 16-point multiple systemic infectious disease syndrome (MSIDS) model was important in achieving remission. In conclusion, DDDCT with one or several 6-7-day pulses of HDDCT, while addressing abnormalities on the 16-point MSIDS map, could represent a novel effective clinical and anti-infective strategy in CLD/PTLDS and associated co-infections including Bartonella.

Comparison of the Efficacy of Longer versus Shorter Pulsed High Dose Dapsone Combination Therapy in the Treatment of Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome with Bartonellosis and Associated Coinfections.

Twenty-five patients with relapsing and remitting Borreliosis, Babesiosis, and bartonellosis despite extended anti-infective therapy were prescribed double-dose dapsone combination therapy (DDDCT), followed by one or several courses of High Dose Dapsone Combination Therapy (HDDCT). A retrospective chart review of these 25 patients undergoing DDDCT therapy and HDDCT demonstrated that 100% improved their tick-borne symptoms, and patients completing 6-7 day pulses of HDDCT had superior levels of improvement versus 4-day pulses if Bartonella was present. At the completion of treatment, 7/23 (30.5%) who completed 8 weeks of DDDCT followed by a 5-7 day pulse of HDDCT remained in remission for 3-9 months, and 3/23 patients (13%) who recently finished treatment were 1 ½ months in full remission. In conclusion, DDDCT followed by 6-7 day pulses of HDDCT could represent a novel, effective anti-infective strategy in chronic Lyme disease/Post Treatment Lyme Disease Syndrome (PTLDS) and associated co-infections, including Bartonella, especially in individuals who have failed standard antibiotic protocols.

Efficacy of Short-Term High Dose Pulsed Dapsone Combination Therapy in the Treatment of Chronic Lyme Disease/Post-Treatment Lyme Disease Syndrome (PTLDS) and Associated Co-Infections: A Report of Three Cases and Literature Review.

Lyme disease and associated co-infections are increasing worldwide and approximately 20% of individuals develop chronic Lyme disease (CLD)/Post-Treatment Lyme Disease Syndrome (PTLDS) despite early antibiotics. A seven- to eight-week protocol of double dose dapsone combination therapy (DDDCT) for CLD/PTLDS results in symptom remission in approximately 50% of patients for one year or longer, with published culture studies indicating higher doses of dapsone demonstrate efficacy against resistant biofilm forms of Borrelia burgdorferi. The purpose of this study was, therefore, to evaluate higher doses of dapsone in the treatment of resistant CLD/PTLDS and associated co-infections. A total of 25 patients with a history of Lyme and associated co-infections, most of whom had ongoing symptoms despite several courses of DDDCT, took one or more courses of high dose pulsed dapsone combination therapy (200 mg dapsone × 3-4 days and/or 200 mg BID × 4 days), depending on persistent symptoms. The majority of patients noticed sustained improvement in eight major Lyme symptoms, including fatigue, pain, headaches, neuropathy, insomnia, cognition, and sweating, where dapsone dosage, not just the treatment length, positively affected outcomes. High dose pulsed dapsone combination therapy may represent a novel therapeutic approach for the treatment of resistant CLD/PTLDS, and should be confirmed in randomized, controlled clinical trials.

Efficacy of Double-Dose Dapsone Combination Therapy in the Treatment of Chronic Lyme Disease/Post-Treatment Lyme Disease Syndrome (PTLDS) and Associated Co-infections: A Report of Three Cases and Retrospective Chart Review.

Three patients with multi-year histories of relapsing and remitting Lyme disease and associated co-infections despite extended antibiotic therapy were each given double-dose dapsone combination therapy (DDD CT) for a total of 7-8 weeks. At the completion of therapy, all three patients' major Lyme symptoms remained in remission for a period of 25-30 months. A retrospective chart review of 37 additional patients undergoing DDD CT therapy (40 patients in total) was also performed, which demonstrated tick-borne symptom improvements in 98% of patients, with 45% remaining in remission for 1 year or longer. In conclusion, double-dose dapsone therapy could represent a novel and effective anti-infective strategy in chronic Lyme disease/ post-treatment Lyme disease syndrome (PTLDS), especially in those individuals who have failed regular dose dapsone combination therapy (DDS CT) or standard antibiotic protocols. A randomized, blinded, placebo-controlled trial is warranted to evaluate the efficacy of DDD CT in those individuals with chronic Lyme disease/PTLDS.

Efficacy of glutathione therapy in relieving dyspnea associated with COVID-19 pneumonia: A report of 2 cases.

PURPOSE: Infection with COVID-19 potentially can result in severe outcomes and death from "cytokine storm syndrome", resulting in novel coronavirus pneumonia (NCP) with severe dyspnea, acute respiratory distress syndrome (ARDS), fulminant myocarditis and multiorgan dysfunction with or without disseminated intravascular coagulation. No published treatment to date has been shown to adequately control the inflammation and respiratory symptoms associated with COVID-19, apart from oxygen therapy and assisted ventilation. We evaluated the effects of using high dose oral and/or IV glutathione in the treatment of 2 patients with dyspnea secondary to COVID-19 pneumonia. METHODS: Two patients living in New York City (NYC) with a history of Lyme and tick-borne co-infections experienced a cough and dyspnea and demonstrated radiological findings consistent with novel coronavirus pneumonia (NCP). A trial of 2 g of PO or IV glutathione was used in both patients and improved their dyspnea within 1 h of use. Repeated use of both 2000 mg of PO and IV glutathione was effective in further relieving respiratory symptoms. CONCLUSION: Oral and IV glutathione, glutathione precursors (N-acetyl-cysteine) and alpha lipoic acid may represent a novel treatment approach for blocking NF-κB and addressing "cytokine storm syndrome" and respiratory distress in patients with COVID-19 pneumonia.

Three novel prevention, diagnostic, and treatment options for COVID-19 urgently necessitating controlled randomized trials.

PURPOSE: Asymptomatic or minimally symptomatic infection with COVID-19 can result in silent transmission to large numbers of individuals, resulting in expansion of the pandemic with a global increase in morbidity and mortality. New ways of screening the general population for COVID-19 are urgently needed along with novel effective prevention and treatment strategies. HYPOTHESIS: A hypothetical three-part prevention, diagnostic, and treatment approach based on an up-to-date scientific literature review for COVID-19 is proposed. Regarding diagnosis, a validated screening questionnaire and digital app for COVID-19 could help identify individuals who are at risk of transmitting the disease, as well as those at highest risk for poor clinical outcomes. Global implementation and online tracking of vital signs and scored questionnaires that are statistically validated would help health authorities properly allocate essential health care resources to test and isolate those at highest risk for transmission and poor outcomes. Second, regarding prevention, no validated protocols except for physical distancing, hand washing, and isolation exist, and recently ivermectin has been published to have anti-viral properties against COVID-19. A randomized trial of ivermectin, and/or nutraceuticals that have been published to support immune function including glutathione, vitamin C, zinc, and immunomodulatory supplements (3,6 Beta glucan) could be beneficial in preventing transmission or lessening symptomatology but requires statistical validation. Third, concerning treatment, COVID-19 induced inflammation and "cytokine storm syndrome" with hemophagocytic lymphohistiocytosis (HLH)/Macrophage Activation Syndrome (MAS) have resulted in extreme morbidity and mortality in those with certain comorbidities, secondary to "acute respiratory distress syndrome" (ARDS) and multiorgan dysfunction with disseminated intravascular coagulation (DIC). Deficiency in red blood cell, serum and alveolar glutathione has been published in the medical literature for ARDS, as well as viral and bacterial pneumonias, resulting from increased levels of free radical/oxidative stress. A randomized controlled trial of blocking NF-κB and cytokine formation using glutathione precursors (N-acetyl-cysteine [NAC] and alpha lipoic acid) and PO/IV glutathione with associated anti-viral effects should be performed, along with an evaluation of Nrf2 activators (curcumin, sulforaphane glucosinolate) which have been scientifically proven to lower inflammation. Since high mortality rates from sepsis induced DIC due to COVID-19 infection has also been associated with thrombotic events and elevated levels of D-dimer, randomized controlled trials of using anticoagulant therapy with heparin is urgently required. This is especially important in patients on ventilators who have met certain sepsis induced coagulopathy (SIC) criteria. The use of acetazolamide with or without sildenafil also needs to be explored with or without heparin, since increased oxygen delivery to vital organs through prevention of thrombosis/pulmonary emboli along with carbonic anhydrase inhibition may help increase oxygenation and prevent adverse clinical outcomes. CONCLUSION AND IMPLICATIONS: A three-part prevention, diagnostic, and treatment plan is proposed for addressing the severe complications of COVID-19. Digital monitoring of symptoms to clinically diagnose early exposure and response to treatment; prevention with ivermectin as well as nutritional therapies that support a healthy immune response; treatment with anti-inflammatory therapies that block NF-κB and activate Nrf2 pathways, as well as novel therapies that address COVID-19 pneumonia and ARDS with DIC including anticoagulation and/or novel respiratory therapies with or without acetazolamide and sildenafil. These three broad-based interventions urgently need to be subjected to randomized, controlled trials.

Effect of dapsone alone and in combination with intracellular antibiotics against the biofilm form of B. burgdorferi.

OBJECTIVE: Lyme disease is a tick-borne, multisystemic disease caused by Borrelia burgdorferi. Standard treatments for early Lyme disease include short courses of oral antibiotics but relapses often occur after discontinuation of treatment. Several studies have suggested that ongoing symptoms may be due to a highly antibiotic resistant form of B. burgdorferi called biofilms. Our recent clinical study reported the successful use of an intracellular mycobacterium persister drug used in treating leprosy, diaminodiphenyl sulfone (dapsone), in combination therapy for the treatment of Lyme disease. In this in vitro study, we evaluated the effectiveness of dapsone individually and in combination with cefuroxime and/or other antibiotics with intracellular activity including doxycycline, rifampin, and azithromycin against Borrelia biofilm forms utilizing crystal violet biofilm mass, and dimethyl methylene blue glycosaminoglycan assays combined with Live/Dead fluorescent microscopy analyses. RESULTS: Dapsone, alone or in various combinations with doxycycline, rifampin and azithromycin produced a significant reduction in the mass and protective glycosaminoglycan layer and overall viability of B. burgdorferi biofilm forms. This in vitro study strongly suggests that dapsone combination therapy could represent a novel and effective treatment option against the biofilm form of B. burgdorferi.

Precision medicine: retrospective chart review and data analysis of 200 patients on dapsone combination therapy for chronic Lyme disease/post-treatment Lyme disease syndrome: part 1.

PURPOSE: We collected data from an online survey of 200 of our patients, which evaluated the efficacy of dapsone (diaminodiphenyl sulfone, ie, DDS) combined with other antibiotics and agents that disrupt biofilms for the treatment of chronic Lyme disease/post-treatment Lyme disease syndrome (PTLDS). We also collected aggregate data from direct retrospective chart review, including laboratory testing for Lyme, other infections, and associated tick-borne coinfections. This helped us to determine the frequency of exposure to other infections/coinfections among a cohort of chronically ill Lyme patients, evaluate the efficacy of newer "persister" drug regimens like DDS, and determine how other infections and tick-borne coinfections may be contributing to the burden of chronic illness leading to resistant symptomatology. PATIENTS AND METHODS: A total of 200 adult patients recruited from a specialized Lyme disease medical practice had been ill for at least 1 year. We regularly monitored laboratory values and participants' symptom severity, and the patients completed the online symptom questionnaire both before beginning treatment and after 6 months on DDS combination therapy (DDS CT). Paired-samples t-tests and Wilcoxon signed-rank nonparametric test were performed on each of eight major Lyme symptoms, both before DDS CT and after 6 months of therapy. RESULTS: DDS CT statistically improved the eight major Lyme symptoms. We found multiple species of intracellular bacteria including rickettsia, Bartonella, Mycoplasma, Chlamydia, Tularemia, and Brucella contributing to the burden of illness and a high prevalence of Babesia complicating management with probable geographic spread of Babesia WA1/duncani to the Northeast. Borrelia, Bartonella, and Mycoplasma species, as well as Babesia microti had variable manifestations and diverse seroreactivity, with evidence of persistence despite commonly prescribed courses of anti-infective therapies. Occasional reactivation of viral infections including human herpes virus 6 was also seen in immunocompromised individuals. CONCLUSION: DDS CT decreased eight major Lyme symptoms severity and improved treatment outcomes among patients with chronic Lyme disease/PTLDS and associated coinfections.

Dopamine-dependent, swimming-induced paralysis arises as a consequence of loss of function mutations in the RUNX transcription factor RNT-1.

Dopamine (DA) is a neurotransmitter with actions across phylogeny that modulate core behaviors such as motor activity, reward, attention, and cognition. Perturbed DA signaling in humans is associated with multiple disorders, including addiction, ADHD, schizophrenia, and Parkinson's disease. The presynaptic DA transporter exerts powerful control on DA signaling by efficient clearance of the neurotransmitter following release. As in vertebrates, Caenorhabditis elegans DAT (DAT-1) constrains DA signaling and loss of function mutations in the dat-1 gene result in slowed crawling on solid media and swimming-induced paralysis (Swip) in water. Previously, we identified a mutant line, vt34, that exhibits robust DA-dependent Swip. vt34 exhibits biochemical and behavioral phenotypes consistent with reduced DAT-1 function though vt34; dat-1 double mutants exhibit an enhanced Swip phenotype, suggesting contributions of the vt34-associated mutation to additional mechanisms that lead to excess DA signaling. SNP mapping and whole genome sequencing of vt34 identified the site of the molecular lesion in the gene B0412.2 that encodes the Runx transcription factor ortholog RNT-1. Unlike dat-1 animals, but similar to other loss of function rnt-1 mutants, vt34 exhibits altered male tail morphology and reduced body size. Deletion mutations in both rnt-1 and the bro-1 gene, which encodes a RNT-1 binding partner also exhibit Swip. Both vt34 and rnt-1 mutations exhibit reduced levels of dat-1 mRNA as well as the tyrosine hydroxylase ortholog cat-2. Although reporter studies indicate that rnt-1 is expressed in DA neurons, its re-expression in DA neurons of vt34 animals fails to fully rescue Swip. Moreover, as shown for vt34, rnt-1 mutation exhibits additivity with dat-1 in generating Swip, as do rnt-1 and bro-1 mutations, and vt34 exhibits altered capacity for acetylcholine signaling at the neuromuscular junction. Together, these findings identify a novel role for rnt-1 in limiting DA neurotransmission and suggest that loss of RNT-1 may disrupt function of both DA neurons and body wall muscle to drive Swip.

Precision Medicine: The Role of the MSIDS Model in Defining, Diagnosing, and Treating Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome and Other Chronic Illness: Part 2.

We present a precision medical perspective to assist in the definition, diagnosis, and management of Post Treatment Lyme Disease Syndrome (PTLDS)/chronic Lyme disease. PTLDS represents a small subset of patients treated for an erythema migrans (EM) rash with persistent or recurrent symptoms and functional decline. The larger population with chronic Lyme disease is less understood and well defined. Multiple Systemic Infectious Disease Syndrome (MSIDS) is a multifactorial model for treating chronic disease(s), which identifies up to 16 overlapping sources of inflammation and their downstream effects. A patient symptom survey and a retrospective chart review of 200 patients was therefore performed on those patients with chronic Lyme disease/PTLDS to identify those variables on the MSIDS model with the greatest potential effect on regaining health. Results indicate that dapsone combination therapy decreased the severity of eight major Lyme symptoms, and multiple sources of inflammation (other infections, immune dysfunction, autoimmunity, food allergies/sensitivities, leaky gut, mineral deficiencies, environmental toxins with detoxification problems, and sleep disorders) along with downstream effects of inflammation may all affect chronic symptomatology. In part two of our observational study and review paper, we postulate that the use of this model can represent an important and needed paradigm shift in the diagnosis and treatment of chronic disease.

Improvement of common variable immunodeficiency using embryonic stem cell therapy in a patient with lyme disease: a clinical case report.

Bone marrow transplantation and stem cell therapies have been used for the treatment of common variable immunodeficiency (CVID) and other life-threatening medical disorders. This is the first known case report in the medical literature describing improvement of both Lyme disease and CVID with human embryonic stem cell therapy.

Empirical validation of the Horowitz Multiple Systemic Infectious Disease Syndrome Questionnaire for suspected Lyme disease.

PURPOSE: Lyme disease is spreading worldwide, with multiple Borrelia species causing a broad range of clinical symptoms that mimic other illnesses. A validated Lyme disease screening questionnaire would be clinically useful for both providers and patients. Three studies evaluated such a screening tool, namely the Horowitz Multiple Systemic Infectious Disease Syndrome (MSIDS) Questionnaire. The purpose was to see if the questionnaire could accurately distinguish between Lyme patients and healthy individuals. METHODS: Study 1 examined the construct validity of the scale examining its factor structure and reliability of the questionnaire among 537 individuals being treated for Lyme disease. Study 2 involved an online sample of 999 participants, who self-identified as either healthy (N=217) or suffering from Lyme now (N=782) who completed the Horowitz MSIDS Questionnaire (HMQ) along with an outdoor activity survey. We examined convergent validity among components of the scale and evaluated discriminant validity with the Big Five personality characteristics. The third study compared a sample of 236 patients with confirmed Lyme disease with an online sample of 568 healthy individuals. RESULTS: Factor analysis results identified six underlying latent dimensions; four of these overlapped with critical symptoms identified by Horowitz - neuropathy, cognitive dysfunction, musculoskeletal pain, and fatigue. The HMQ showed acceptable levels of internal reliability using Cronbach's coefficient alpha and exhibited evidence of convergent and divergent validity. Components of the HMQ correlated more highly with each other than with unrelated traits. DISCUSSION: The results consistently demonstrated that the HMQ accurately differentiated those with Lyme disease from healthy individuals. Three migratory pain survey items (persistent muscular pain, arthritic pain, and nerve pain/paresthesias) robustly identified individuals with verified Lyme disease. The results support the use of the HMQ as a valid, efficient, and low-cost screening tool for medical practitioners to decide if additional testing is warranted to distinguish between Lyme disease and other illnesses.