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.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  Borrelia burgdorferi (Bb), is a rapidly growing healthcare problem across the globe. In 2022, the United States (US) had an estimated 476,000 new diagnosed cases of Lyme disease (LD) , vitamin and mineral deficiencies, and sleep disorders) [The most frequent scientific hypotheses to explain chronic persistent symptomatology in those suffering from CLD/PTLDS involve multiple mechanisms, including not only co-infections ,38, but Borrelia ,46,47,48Borrelia . HorowitBorrelia . These isorders) ,59,60,61sorders) , hormonasorders) ,64, Postsorders) , autoimmsorders) ,66, neursorders) , liver dsorders) ,69 and rsorders) . Patientsorders) .Borrelia burgdorferi as a potential cause of persistent infection and ongoing inflammation that could account at least in part for continued symptoms in CLD/PTLDS [Borrelia persists because of a combination of factors. These include immune evasion and changing outer surface proteins [Borrelia [In the past several years, researchers at Johns Hopkins University, the University of New Haven, Stanford University, and Northeastern University have identified stationary, persister, and biofilm forms of LD/PTLDS ,73,74,75LD/PTLDS  and a siLD/PTLDS . Previouproteins ,79, resiproteins  with redproteins , persistproteins , macrophproteins , fibroblproteins ,84, and proteins , changinproteins ,86, resuproteins , as wellproteins . The sciBorrelia  and the Borrelia  may accoBorrelia , as biofBorrelia ,93.Babesia and Bartonella: disulfiram (DSF) and dapsone, diaminodiphenylsulphone (DDS) [Bartonella henselae, and 83.3% reported some clinical improvement, although only two patients achieved full remission with high-dose DSF alone (16.7%), while several patients on either minocycline or azithromycin with rifampin and DSF did not achieve remission [Two repurposed medications have been published to have effects on the persister/biofilm forms of Bb ,75 and ane (DDS) ,95. In ane (DDS) . One patne (DDS) . In anotne (DDS) . Twelve emission . The smaB. burgdorferi biofilm. The 4-drug combo of dapsone, a tetracycline, rifampin, and macrolide (azithromycin) had the most significant effect [Dapsone therapy, using dapsone alone, with a tetracycline and/or rifampin, or with or without azithromycin, has also been shown to be effective against the biofilm/persister forms of Bb . Althougt effect .Bartonella henselae, a facultative intracellular gram-negative bacteria, has been found to have stationary biofilm/persister forms [Bartonella species, apart from B. henselae, have been published to establish long-term stealth infections in immunocompetent persons, including B. vinsonii subsp. berkhoffii, B. bacilliformis, and B. quintana, leading to a chronic, persistent infectious state [B. henselae in culture, methylene blue and rifampin were the most active agents against the biofilm B. henselae after 6 days of drug exposure, whereas both rifampin and methylene blue and azithromycin and methylene blue completely eradicated the biofilm form of B. henselae in culture after treatment for 6 days [Babesia, and chronic bartonellosis, with variable results [Similarly, er forms , accounter forms . Other Bus state . Among 1r 6 days . To date results ,103.Bartonella titers turned positive in this patient after adding pyrazinamide (PZA) to other intracellular antibiotics , where Behçet’s ulcers and granulomatous skin changes resolved for the first time in decades, implicating a potential role for Bartonella in the autoimmune manifestations of Behçet’s [In five separate clinical studies involving more than 365 patients, dapsone combination therapy led to statistically verified improvement in relieving eight major Lyme symptoms in our patients, including sweats/chills, fatigue, joint, muscle, and/or nerve pain, headaches, insomnia, and cognition ,103,104.Behçet’s . erythema migrans (EM) rashes, 100% improved, and 58% went into long-term remission post-DDDCT [Bartonella RNA in their blood and were FISH-positive achieved long-term remission post-treatment with DDDCT. Among 11 patients with a history of Bartonella exposure and no evidence of active infection , 5 (45%) remained in remission, and 6 (55%) improved their underlying symptomatology [Babesia and Bartonella. Of 19 patients (76%) with a history of Bartonella exposure, 15/19 improved (79%), and among 8 patients with proof of active Bartonella infection, i.e., PCR positive, FISH positive, and/or elevated vascular endothelial growth factor (VEGF), 3/8 (38%) remained in remission, with two out of four who were Bartonella FISH positive going into remission (50%). The conclusion of the study was that the higher dapsone dosage, not just the treatment length, positively affected outcomes [A common clinical finding in these dapsone studies for CLD/PTLDS and associated co-infections, similar to studies using DSF, is that higher doses of dapsone showed superior efficacy in putting patients into long-term remission. Double-dose dapsone combination therapy (DDSCT) using dapsone 100 mg PO BID with a tetracycline and rifampin was superior to single-dose dapsone (100 mg QD) with a tetracycline and rifampin, leading to tick-borne symptom improvements in 98% of patients, with 45% remaining in remission for 1 year or longer . In thosst-DDDCT  if all Mmatology . When himatology . A totaloutcomes . Bartonella persisters [Bartonella and Babesia achieved remission after 8 weeks of DDDCT and 4 days of HDDCT. This left us with several essential research questions. We knew that in culture 6 day of treatment with rifampin and/or azithromycin plus methylene blue eliminated Bartonella persisters. Taking these results from “bench to bedside”, would a longer 6–7 day pulse of HDDCT with higher doses of MB result in superior clinical efficacy against the most common co-infection preventing the long-term success of our DDDCT protocol, Bartonella? Would higher doses of methylene blue show increased efficacy against Lyme persisters, based on its identification from an FDA drug library as having anti-persister activity against Borrelia burgdorferi [Bartonella, improve long-term outcomes? Would higher doses of methylene blue as well as higher doses of folic acid improve the tolerance of DDDCT and HDDCT by reducing dapsone-induced anemia and its inhibition of the folic acid pathway while further lowering methemoglobin levels, improving tolerability [Babesia and Bartonella). Among 23 patients who completed a full 8-week DDDCT protocol prior to at least one 4-day or 5–7 day HDDCT pulse, 7/23 (30.4%) went into full remission for 3-months or longer, and 3/23 (14%) went into remission for 6 weeks or longer. Methylene blue (MB) was used in both the double dose and pulsed high dose dapsone studies to help lower methemoglobin levels, a known side effect of dapsone , at dosersisters ,101. Onlgdorferi , apart fgdorferi ? Would orability ,110? In We closely examined 25 charts of adult patients who completed the DDDCT and HDDCT protocols.A total of 19 of the 25 patients (76%) had previously completed one course of DDDCT; 2 patients (8%) completed 2 courses of DDDCT; and 2 patients (8%) completed 3 courses of DDDCT before doing one or several courses of HDDCT, which consisted of either a short pulse (4 days) or a longer pulse (5–7 days). Three patients in this chart review had previously undergone one or more treatments with disulfiram and/or DDDCT and had either failed or had an inadequate response to prior antibiotic therapy and/or had relapsed with persistent symptoms after stopping anti-infective therapy. Three patients were also unable to complete the protocol as prescribed. Two patients (8%) did not complete a full 28-day course of DDDCT prior to completing a HDDCT pulse. One was due to a severe Herxheimer flare with neuropsychiatric symptoms secondary to DSF use with DDS, and another was due to viral gastroenteritis. Another patient was unable to complete the 6-day HDDCT pulse as prescribed with 300 mg of MB due to his history of severe neuropsychiatric symptoms with psychosis, requiring a continuation of his psychiatric medicine. Borrelia-specific bands  on a WB [We assessed co-infection status, age, gender, length of illness, and response to treatment, i.e., self-reported improvement in Lyme symptoms, and whether there was remission, percentage improvement, or lack of response to the HDDCT protocol. Full remission was defined as the resolution of all active tick-borne symptoms for at least 3 months post-therapy. Partial remission was defined as the resolution of all active tick-borne symptoms for at least 6 weeks post-therapy since some patients had recently finished the HDDCT protocol. All 25 patients in our retrospective chart review met the criteria for a clinical diagnosis of Lyme disease supported by a physician-documented erythema migrans (EM) rash and/or positive laboratory testing, including a positive ELISA/enzyme immunoassay (and/or C6 ELISA), immunofluorescent antibody (IFA), Centers for Disease Control and Prevention (CDC) positive IgM and/or IgG Western Blot (WB), PCR,  on a WB ,112, andSaccharomyces boulardii, were used to help prevent antibiotic-associated diarrhea [All patients signed informed consent forms that outlined the proposed benefits and the potential risks of treatment. Patients volunteered to take the high-dose pulsed dapsone protocol at our medical practice based on our prior research illustrating the benefit of dapsone combination therapy in the treatment of CLD/PTLDS ,103,104 diarrhea ,124.In order to minimize potential drug interactions with methylene blue, including serotonin syndrome/toxicity , patientDietary restrictions were also instituted, avoiding foods high in tyramine, which can potentially produce a hypertensive crisis in the presence of MAOIs and/or higher doses of methylene blue . PatientBorrelia [Several patients took disulfiram (DSF) in combination with HDDCT if they had previously failed to have an adequate clinical improvement with either drug regimen used alone or in combination, utilizing higher dosing if they failed prior combination therapies. The patients signed a consent informing them of the potential benefits and risks of DSF, including increased fatigue, brain fog/cognitive dysfunction, worsening psychiatric symptoms, liver function abnormalities, and/or increased neuropathy ,97. TheyBorrelia , along wBorrelia . All patBorrelia .Borrelia burgdorferi [Borrelia [Although both dapsone and disulfiram have demonstrated some efficacy against resistant biofilm/persister forms of gdorferi ,94,104, gdorferi , Biocidigdorferi , essentigdorferi , as wellgdorferi . Rarely,gdorferi , was congdorferi . If patigdorferi ,140. GraBorrelia ,142.Institutional Review Board approval was not required for this work since this was a retrospective review of a convenience sample of patient charts. A convenience sample of 25 adult patient charts was chosen for inclusion in our study out of a patient population of 50 patients offered HDDCT. Protocol 1 with Protocol 1. Medication and Nutritional Supplementation Protocol Sheet for Double Dose Dapsone Combination Therapy and High Dose Pulsed Dapsone Combination Therapy for CLD/PTLDS.Diet: Avoid aged cheese, liver, avocados, beer, wine, overripe fruit, raisins, sauerkraut, sour cream, soy sauce, yeast extracts, etc. since a low histamine diet will help protect against any increases in blood pressure while on higher doses of methylene blue (MB). Those with high blood pressure (hypertension) should have a home electronic blood pressure cuff and intermittently check their blood pressure while taking MB. Stay on a low-carbohydrate diet and avoid simple carbohydrates to avoid promoting yeast/candida overgrowth. Take tetracyclines with a full stomach and 8 oz glass of water, sitting up for one hour post-ingestion to avoid reflux esophagitis; do not mix dairy and minerals within one hour of use; and avoid more than several minutes of direct sun on tetracyclines  since they can cause a sunburn. Use of a 65-sun protection factor sunscreen on all exposed areas  is advisable, even while driving in the car.The patient should be Glucose-6-Phosphate Dehydrogenase (G6PD) positive (an enzyme needed to help red blood cells work properly) without significant iron deficiency anemia or vitamin deficiency  before starting this protocol. Moreover, before instituting methylene blue, all psychiatric medications that could interact should be tapered and stopped (except benzodiazepines). The patient should provide a list of all medications and supplements for the healthcare provider to review, as medication interactions can occur  and may require a dosage adjustment in medications . Coordination with a psychiatrist may be required, depending on the number and types of psychiatric medications. Protocol 2. Two-week HDDCT Protocol Medication and Nutritional Supplementation Sheet for CLD/PTLDS.Bartonella). This protocol is primarily for patients who have completed an 8-week DDDCT in the past (months or years) with or without a course of HDDCT and have ongoing resistant symptoms attributable to tick-borne disease  had been ill for more than 20 years, 40% (n = 10) had been ill between 10 and 20 years, 24% (N = 6) had been ill between 5 and 9 years, and 12% (n =3) had been ill for less than 5 years in duration. Eight percent of patients (N = 2) had physician-diagnosed EM rashes and therefore met the criteria for PTLDS. Another patient had multiple red rashes 22 years ago, prior to getting ill; however, the diagnosis of Lyme disease had not been established by a physician. Among these 25 participants with CLD/PTLDS, 44% (11/25) had CDC-positive IgM Lyme immunoblots, and one patient out of 25 (4%) had at least one co-infection; 2 patients (8%) had at least 2 co-infections; and 22 out of 25 patients (88%) had three or more co-infections. Five patients (20%) had positive titers for Relapsing Fever Borrelia (TBRF), 3 patients (12%) had antibody titers to Borrelia miyamotoi, and 16/25 patients (64%) had exposure to Babesia species, with several patients having more than one species present ; 1 Babesia divergens). Five out of 25 patients (20%) had evidence of active Babesia, as they were Babesia FISH positive. Twenty-one patients (84%) tested positive for one or more Bartonella species. These included: 11 patients with Bartonella henselae; 7 with Bartonella quintana; 6 with Bartonella elizabethae; 6 with Bartonella vinsonii subspecies; 1 with Bartonella koehlerae; 1 with Bartonella bacciliformis; and 4 with Bartonella spp. (genus) without specific spp. identification. Seventeen patients (68%) had evidence of active Bartonella, with 11 patients (44%) being positive by Bartonella FISH and 7/25 (28%) having evidence of active Bartonella via elevated vascular endothelial growth factor (VEGF) or changes in striae. Other co-infections included 20% of patients with evidence of Ehrlichia (5/25), 16% (4/25) with Anaplasma, 24% (6/25) with low positive tularemia titers, 16% (4/25) with Rickettsia spp., 16% (4/25) with low positive titers to Brucella spp., 72% (18/25) with prior exposure to Mycoplasma pneumonia, 12% (3/25) with Chlamydia pneumonia, 96% (24/25) with evidence of exposure to herpes viruses 1, 2, 6, and EBV ; 20% (5/25) with Cytomegalovirus (CMV); 8% (2/25) with West Nile virus (WNV) exposure; 52% (13/25) with COVID exposure before, during, or after DDDCT and HDDCT; 32% (8/25) were positive to Coxsackie/Parvovirus B-19; and 60% (15/25) had evidence of Candida/hypoglycemia.Among the 25 participants, 15 were men and 10 were women. The age range was between 18 and 63 years old . A total of 68% were less than 50 years old, and 32% were older than 50 years old. The mean length of illness was 14.7 years. A total of 24% of patients ; Trichothecenes, 11/25 (44%); Aflatoxins, 8/25 (32%); Gliotoxins, 11/25 (44%); Zearalenone, 9/25 (36%); Citrinin/Dihydrocitinone, 1/25 (4%); exposure to 8 heavy metals (Mercury (Hg) 10/25 patients (40%); Lead (Pb), 11/25 (44%); Arsenic (As), 3/25 (12%); Tin (Sn), 1/25 (4%); other: Cesium (Ce), Uranium (Ur), Thallium , Antimony (Sb), 7/25 (28%); Inflammation/Autoimmune markers: 20/25 (80%); Immune Deficiency (CVID), 5/25 (20%); Other immune abnormalities  cells, ↑ IgM antibodies), 5/25 (20%); Leaky Gut/Food Sensitivity/Mast Cell Activation Syndrome (MCAS), 15/25 (60%): GI Microbiome Abnormalities, Gastroparesis, 6/25 (24%); Liver Abnormalities, i.e., Non-alcoholic Steatohepatitis (NASH), ↑ LFT’s, 11/25 (44%); Liver Abnormalities, Low Alkaline Phosphatase, 6/25 (24%); Detoxification Abnormalities, Low glutathione (GSH), 7/25 (28%); Vitamin and mineral deficiencies, 22/25 (88%); Hormonal abnormalities, low adrenal function, 20/25 (80%); Hormonal abnormalities, low testosterone (men), 6/25 (24%); Hormonal abnormalities, absent pituitary function, 1/25 (4%); Hormonal abnormalities, elevated insulin/Polycystic Ovarian Syndrome (PCOS), 1/25 (4%), Hormonal abnormalities, low thyroid function, 7/25 (28%); insomnia/sleep disorders , 17/25 (68%); POTS/dysautonomia, 8/25 (32%); Psychiatric disorders , PTSD, Schizophrenia), 14/25 (56%); Mitochondrial dysfunction, 6/25 (24%); and genetic abnormalities , 10/25 patients (40%). Babesia and/or Bartonella. Three out of 23 patients (13%) also had a full resolution of their Lyme and tick-borne symptoms for 1½ months or longer, having recently completed the full protocol 6 weeks prior; among those three patients, patient number 10  had improvement in their tick-borne symptoms. Of 23 patients who completed a full 8-week course of DDDCT prior to one or several courses of HDDCT, 7/23 (30.5%) had a full resolution of their Lyme and tick-borne symptoms for 3 months or longer, even if there was prior evidence of co-infections, including A total of 2/25 patients (8%) were EM rash positive with a history of PTLDS, and 1/2 (50%) went into remission for 3 months or longer. Patient 5 had an EM rash 21 years prior, and although DDDCT in 2019 helped to relieve the vast majority of symptoms for several years, it was not until completing a 4-day course of HDDCT 3 years later, in December 2022, that residual fatigue, joint pain, irritability, insomnia, and word recall issues resolved 100% for 6 months or longer.Babesia FISH positive remained in remission, although 4/6 of them (66.6%) were greater than 30% improved and 2 patients were somewhat improved (range of 0–20%). The last patient, patient number 25 (see Of the sixty-four percent (16/25) of patients with babesiosis, 18.8% (3/16) were in full remission. None of the 6 patients who were r 25 see , was funBabesia. It was well tolerated in 5/6 patients (83.3%), and DSF needed to be stopped in one patient due to a neuropsychiatric flare with anxiety. Disulfiram was taken with dapsone for the treatment of resistant Babesia in patient number 4 (B. duncani), patient 13 , patient 17 , patient 18 (Babesia FISH+), patient 21 (Babesia duncani), and patient 25 (B. duncani), and the combination therapy was helpful in relieving symptoms of babesiosis, i.e., symptoms of sweats, chills, flushing, air hunger, and/or a cough. Regimens for Babesia apart from using dapsone and DSF included rotations of atovaquone with or without azithromycin and/or clindamycin [Artemisia annua) [Cryptolepis sanguinolenta) [Scutellaria baicalensis) and Japanese knotweed (Polygonum cuspidatum) [Babesia symptoms, except for patient 21. None of the patients who had active Babesia (FISH+) who took dapsone and DSF with or without the above antimalarial medications and botanicals, however, remained in full remission.Six out of 25 patients (24%) took DSF in doses varying between 62.5 mg alternating with 125 mg, up to 500 mg per day, for up to 6 months at a time, during their course of therapy with dapsone for the treatment of Lyme disease and ndamycin ,148, atondamycin , tafenoqndamycin , with orndamycin  and Malandamycin , low dosndamycin , and bota annua) , with ornolenta) , as wellpidatum) . Five ouBartonella exposure, 100% improved their underlying symptomatology after at least one course of DDDCT and one or several courses of HDDCT pulses (4 days or 5–7 days). Four out of 21 patients (19%) went into long-term remission for 3 months or longer; 3/21 patients (14.3%) went into full remission for up to 6 weeks; and 14/21 patients (66.6%) improved. Of those patients not in full remission, symptom improvement for all Bartonella patients was at levels ranging from 15% to greater than 70%. Examining the differences in treatment response between a 4 day pulse and a 5–7 day HDDCT pulse for the treatment of Bartonella, 6/21 patients (28.5%) who took at least one course of DDDCT and one 5–7 day HDDCT pulse went into remission, and no patients who took DDDCT with one or several 4 day HDDCT pulses went into remission if Bartonella was active. An 8-week DDDCT followed by a 5–7-day HDDCT pulse was therefore superior to a shorter 4-day HDDCT pulse for those with CLD who also had chronic active Bartonella. Among the 8 patients with CLD and Bartonella who took one or several 4-day HDDCT pulses without a 5–7 day HDDCT pulse, there was nevertheless significant improvement in underlying symptomatology. Symptoms of bartonellosis that improved with either DDDCT followed by 4 day or 5–7 days HDDCT pulses with MB included 8 major Lyme symptoms, i.e., fatigue, joint pain, myalgias, neuropathy, headaches, sweats, chills, cognitive functioning, mood, and insomnia, but also included improvement in pain in the soles of the feet, striae, and Bartonella pseudo seizures  [Among 21/25 patients (84%) who had a history of eizures) .Bartonella. Three patients who did one course of DDDCT and one course of HDDCT for 4 days  went into long-term remission, and none of them had evidence of exposure to Bartonella. The 4-day HDDCT pulse after 8 weeks of DDDCT was therefore adequate for treating chronic LD/PTLDS if Bartonella was not present. All patients except patient 23 took PZA during DDDCT + HDDCT.An 8-week DDDCT followed by a 4-day HDDCT pulse was, however, effective in putting several patients into long-term remission with CLD/PTLDS if there was no evidence of exposure to Bartonella FISH positive, and patients 14 and 21, who had elevated VEGFs, consistent with active Bartonella. Patient number 10 did her first HDDCT pulse with 100 mg of methylene blue (MB) PO BID, and she was one month symptom-free before relapsing. She therefore did a second 4-day HDDCT pulse, using 200 mg PO BID of MB, which improved symptomatology but was insufficient to put her in full remission. A third HDDCT pulse for 4 days with 300 mg PO BID of MB was therefore instituted, which resulted in her being symptom-free for 2 months, her longest time without a relapse. She recently finished her first 6-day HDDCT pulse using 300 mg PO BID of MB after doing 3/4-day HDDCT pulses with lower doses of MB and has remained 100% symptom-free (approximately 2 more months in remission). Patient number 24 suffered from Lyme disease (C6 ELISA positive), active Babesia (FISH+), active Bartonella (FISH+), and multiple MSIDS abnormalities, including hypopituitarism, POTS, CVID (on IVIG), small fiber neuropathy, severe resistant insomnia, anxiety with PTSD, inflammation, multiple vitamin and mineral deficiencies, and Morgellons’s disease. She had conducted 2 courses of DDDCT, 3 courses of HDDCT for 4 days, and one course of HDDCT for 6 days. Earlier courses of pulse HDDCT were for 4 days and used a lower dose of MB (100–150 mg PO BID). The last 6-day HDDCT pulse used 300 mg of MB. Although there was temporary improvement in symptomatology with DDDCT and three four-day pulses of HDDCT with lower dose MB, it was not until the last course of six days of HDDCT with 300 mg PO BID of MB that she noticed persistent and sustained improvement in energy, myalgias, neuropathy, and cognitive impairment for five months. Patient 21, after 3 courses of DDDCT and 3 courses of HDDDT for 4 days, where the last round used a higher dose of MB at 300 mg BID, resulted in sustained improvements in energy/endurance, headaches, decreased joint pain, decreased neuropathy, dizziness, and some movement of paralyzed muscles of the lower extremities during the past 4 months. The lower doses of MB used in 4-day HDDCT pulses did not result in sustained improvement or movement of paralyzed muscles in the lower extremities. Finally, patient 14 had 3 spp. of Bartonella , an elevated VEGF, implying active Bartonella, and a Bartonella granuloma on her right hand. She also suffered from severe depression and anxiety with POTS, similar to the other patients with multiple species of Bartonella and Babesia. After 2 courses of DDDCT and one 6–7 day course of HDDCT with minocycline, rifampin, azithromycin, and pyrazinamide in December 2022, along with a higher dose of MB at 300 mg PO BID, she noticed a marked improvement in clinical symptomatology, going from 25% to 87% of normal functioning, with improvements in fatigue, chills, joint pains, and cognition. Her joint pain and swelling decreased, and the Bartonella granuloma on her right hand decreased in size by 80%. See Methylene blue (MB) dosage, apart from the length of HDDCT pulses, affected the efficacy and tolerability of the protocol. Earlier regimens of 4-day HDDCT used 100 mg of MB PO BID (twice a day). These doses were subsequently increased after patients relapsed post-therapy, with evidence of increased methemoglobin. The MB dose was therefore increased to 150–200 mg PO BID, and if symptom relapses persisted with elevations in methemoglobin, the dose was increased to a final dose of 300 mg PO BID while using HDDCT. Examples of increased efficacy of higher doses of MB were seen in patients 10 and 24, who were both Age, length of illness (LOI), and gender were also evaluated to determine their effect on the treatment outcome. Neither age, LOI, nor gender affected remission rates or levels of improvement. Among 17 patients less than 50 years of age, 4 patients went into full remission for 3 months or longer; one patient went into remission for 6 weeks or longer; 5 patients improved greater than 50% of normal; 1 patient improved by greater than 45% of normal; 2 patients improved by 30% of normal; 3 patients improved by 15% of normal; and 1 patient improved by 5% of normal. In the 8 patients who were greater than 50 years old, 3 patients went into full remission for 3 months or longer, 2 patients went into full remission for 6 weeks or longer, and 3 patients improved their baseline functioning by greater than 50% of normal. Although there were fewer patients (8 vs. 17) greater than 50 years old, equal numbers (5 patients in each group) went into remission, and all patients had significant improvement.Length of illness and gender also did not affect remission rates or clinical improvement. Sixteen patients (64%) were sick for 10–20 years or longer, and 8/16 (50%) were in remission, with 8/16 (50%) showing significant clinical improvement. Among 5 patients who had been ill for more than 20 years, 1 was in full remission (3 months or longer), 2 were in remission for 6 weeks, one patient had improved by 30%, and another patient had improved by 15%; Eleven patients were sick between 10 and 20 years old, and 5 went into full remission; 4 were improved by greater than 50% of normal; and 2 patients were improved by 15% of normal. Eight patients were sick between 5 and 9 years, and one was in full remission; one was in remission for 6 weeks; 3 improved by 50% or greater; 2 improved between 30 and 45%; and one improved by 5%; and finally, one patient was sick for 0–4 years and was in full remission.Borrelia burgdorferi [Gender also had no effect on clinical improvement or rates of remission. Although there were more men (15) than women (10) who took DDDCT and HDDCT, equal numbers of men and women  were in remission. Among those who did not go into full remission, 60% of women (3/5) and 53.3% of men (8/15) also improved by 30% or greater, with 2 women and 2 men both having improvements ranging from 5–15%. In a recent study by Johnson et al., using self-reported clinical data from 2170 patients in the MyLymeData patient registry , the autgdorferi ,158,159.Babesia spp. and several Bartonella spp. simultaneously, these patients were some of the sickest and most resistant patients. Four out of five of these patients (80%) with multiple species of Babesia and Bartonella (80%) were Bartonella FISH-positive. One patient who was Bartonella FISH+ and had Bartonella striae suffered from psychosis with auditory hallucinations, and clinical characteristics shared among the other patients included severe neuropsychiatric symptoms  as well as resistant neuropathy . Patient 11 had chronic inflammatory demyelinating polyneuropathy, i.e., CIDP on IVIG; patient 21 had small fiber neuropathy and was on IVIG; and patient 23 also had CIDP and small fiber neuropathy. Patients 11, 17, 21, 23, and 24 had resistant neuropathic symptoms of tingling, numbness, burning, or stabbing sensations, and patients 11, 21, and 23 had severe POTS/dysautonomia, a form of autonomic neuropathy. The species of Babesia and Bartonella that were found in these 5 patients , and patients 15 and 23 had evidence of 3 species of Babesia . Patients 11 and 21 had evidence of 2 spp. of Bartonella , patients 12 and 14 had evidence of 3 spp. of Bartonella , and patients 15 and 23 had evidence of 4 spp. of Bartonella . Patients who simultaneously had two or more species of Babesia and Bartonella oftentimes required psychiatric medication to help control their depression/anxiety and medication (gabapentin (Neurontin), amitriptyline (Elavil), pregabalin (Lyrica), and/or duloxetine ) to control neuropathic symptoms [Bartonella (Bh and Bq) with elevated VEGF and low positive tularemia and Brucella titers, which both can cross-react with other intracellular pathogens [Examining the clinical symptomatology and difference in response rates of those patients who had evidence of exposure to several ents see  includedsymptoms ,162,163.symptoms , midodrisymptoms , droxidosymptoms , Beta blsymptoms , and ivasymptoms , she wasathogens ,170.Despite changes in a number of laboratory values during the course of treatment described here, all patient values returned to normal at the end of treatment . Laboratory values with a CBC, CMP , and methemoglobin levels were monitored before, during, and after DDDCT and HDDCT. For some patients, there were temporary changes in white cell counts , platelets (3 patients had mild thrombocytopenia), transient elevation in myelocytes and metamyelocytes (4–5 patients), regular decreases in hemoglobin (Hb) and hematocrit (Hct) and expected rises in the mean corpuscular volume (MCV) while on dapsone, due to the drug’s effect as a folic acid inhibitor, affecting bone marrow production . There wThe details of the changes in laboratory parameters were as follows: White cell counts remained WNL for 84% (21/25) of patients , with a mean WBC count of 5.57 per µL (median 4.75 µL). Four patients (16%) had temporary episodes of leucopenia (minimum 2.36 µL) and one patient had a temporary episode of leukocytosis (maximum 18.5 µL). All WBC values returned within normal limits post therapy. Hemoglobin (Hb) values   were monThe last laboratory parameters we monitored before, during, and after DDDCT and HDDCT were liver functions, including aspartate transaminase , alanine transaminase , alkaline phosphatase , and total bilirubin  ,186. ASTBabesia and Bartonella, with some patients reactivating underlying viral infections . Our cohort of CLD patients also had other overlapping sources of inflammation on the 16-point MSIDS map contributing to their persistent illness, including environmental toxins , microbiome abnormalities, leaky gut with food sensitivities/mast cell activation syndrome (MCAS), sleep disorders, and vitamin and mineral deficiencies [Babesia and active Bartonella played a major contributing role in driving chronic symptomatology. Sixteen out of 25 patients (64%) with CLD/PTLDS had exposure to Babesia spp., and 21/25 patients (84%) tested positive for one or more Bartonella species. The 3 patients with CLD/PTLDS  who went into long-term remission with 8 weeks of DDDCT and one 4-day pulse of HDDCT had no evidence of active Babesia or Bartonella, despite an LOI between 11 and 21 years. None of the 6 patients who had active Babesia by FISH went into full remission, requiring multiple rotations of anti-malarial medications, and those patients who had evidence of exposure to multiple species of Babesia and Bartonella were some of the sickest patients with resistant neuropsychiatric symptoms and peripheral/autonomic neuropathy (POTS). In those Lyme and Bartonella patients who were FISH+ (and/or had an elevated VEGF and/or striae with granulomas) with a history of Babesia exposure (FISH negative), a longer course of HDDCT (5–7 days) was required post-8 weeks of DDDCT to achieve full remission. Seven out of 23 patients (30.5%) with CLD/PTLDS with Bartonella and Babesia who completed 8 weeks of DDDCT followed by a 6 to 7 day pulse of HDDCT remained in remission for a period of 3–9 months, and 3/23 patients (13%) recently completed the protocol and so far have been in remission for at least 1 ½ months. Although 100% of all 25 patients had an improvement in underlying symptomatology with DDDCT and HDDCT, and 43.5% of patients were in remission at the time of submission of this publication, overlapping MSIDS factors interfered with many patients who did not go into full remission. These factors primarily included inflammation/autoimmune phenomena (80%), due to not only chronic infections but also environmental toxins with mold and heavy metals (88%), immune dysfunction with CVID and CIDP (36%), hormonal abnormalities with low adrenal function, low testosterone (low T) (80%), leaky gut/food sensitivities/microbiome abnormalities/MCAS (68%), hypoglycemia/candida (up to 52%), vitamin and mineral deficiencies (88%), detoxification abnormalities including low glutathione (28%), liver abnormalities, including non-alcoholic steatohepatitis (NASH) (56%), resistant insomnia (68%), POTS/dysautonomia (32%), and severe neuropsychiatric symptoms (56%). Establishing a broad differential diagnosis among those suffering from CLD/PTLDS and screening with the 16-point MSIDS map is essential from our perspective, since many of these MSIDS factors can cause resistant fatigue, pain, cognitive difficulties, insomnia, and neuropsychiatric symptoms resembling chronic Lyme and tick-borne infections, yet their treatment and resolution do not involve antibiotic therapy.Lyme disease has reached epidemic proportions in the United States and worldwide, with an estimated global Bb seroprevalence of 14.5% . Althougciencies ,102,103.ciencies ,70,71. WBabesia duncani, and Bartonella vinsonii subspp., had been vaccinated and boosted several times against COVID-19, and contracted COVID-19 seven times during the course of her illness due to her job, which required regular travel across continents. Despite 7 episodes of COVID-19, one course of DDDCT, and 5 courses of 4-day pulses of HDDCT, her health significantly improved, with each pulse of HDDCT removing a resistant symptom. It was not until she did one 7-day pulse of HDDCT with 300 mg of MB for Lyme and Bartonella that she went into full remission for 3 months or longer.Long COVID also has overlapping symptoms resembling CLD/PTLDS, including chronic fatigue, muscle/joint and nerve pain, insomnia, “brain fog” with cognitive difficulties, neuropsychiatric problems, chest pain, shortness of breath, palpitations, and dizziness secondary to POTS . One wayp-value < 0.00001), where DDS decreased the incidence of Alzheimer’s disease exacerbation [Borrelia burgdorferi has been found in the CNS in patients with Alzheimer’s disease [Dapsone (DDS) alone or in combination with other antibiotics (doxycycline) has been proposed as a potential treatment for those with COVID-19 due to the drug’s combined effects in blocking inflammatory storms and suppressing the production of cytokine signatures including IL1α, IL8, IL1β, IL6, and IL8 and tumor necrosis factor-α ,198. DDSerbation . The poserbation . Althoug disease ,207,208,Borrelia after standard courses of antibiotics, increasing inflammation [Borrelia “persisters” in biofilms [Seven of the factors found on the 16-point MSIDS factors in those suffering from CLD/PTLDS due to ongoing inflammation and downstream effects have been reported in the long COVID-19 syndrome, i.e., viral persistence (coronavirus), viral reactivation (EBV), autoimmunity, mitochondrial dysfunction, POTS/dysautonomia, dysbiosis of the gastrointestinal tract, and MCAS ,214,215.ammation . The scibiofilms ,90,217, biofilms  in biofibiofilms ,91,219, biofilms .Borrelia can persist after standard courses of antibiotics [Borrelia has similarly been proven in the animal model [Borrelia. Although 1–2 courses of high-dose DSF have been shown to have some clinical efficacy in patients with CLD and associated co-infections, resulting in remission rates of up to 36.4% with 6–18 months of use [B. burgdorferi persisters was shown to lack efficacy  46.3%) [Borrelia burgdorferi stationary phase persisters in biofilms, both as a single drug and in combination [B. burgdorferi in these sulfa drug combinations” [Borrelia persisters in culture after evaluating dapsone as a single agent or in combination with tetracycline, rifampin, and azithromycin [Borrelia persisters; however, the efficacy against the persister/biofilm forms of Bb increased as antibiotic combinations were added. Dapsone in combination with doxycycline (2-drug combo) was more effective than dapsone alone, and dapsone + doxycycline + rifampin (3-drug combo) was more effective than the 2-drug combination. The most significant effect in reducing the mass and viability as well as the protective mucopolysaccharide layers of B. burgdorferi biofilm was, however, using the 4-drug combination of dapsone + doxycycline + rifampin + azithromycin [Borrelia persisters using multiple antibiotic combinations [There are multiple scientific studies showing that ibiotics ,221,222,ibiotics ,224, ligibiotics , fibroblibiotics ,84, intribiotics , endotheibiotics , skin [2ibiotics ,227, eyeibiotics , and cenibiotics ,230, witibiotics . Persistal model , in miceal model , dogs [2al model , horses al model , and macal model , as wellal model ,235; howal model ,97 and dal model ,103,104.) 46.3%) . Dapsonebination . They conations” . Dr. Evahromycin . The conhromycin . The stuinations . To dateinations , which cinations .Bartonella [Bartonella FISH positive (seven patients) or PCR/biopsy positive (two patients) using lower dose dapsone went into remission [Babesia and Bartonella. The aim of this study was to examine the effect of higher-dose dapsone pulses in the treatment of resistant Lyme and associated co-infections as well as the effect of methylene blue (MB). In that study, using a 4-day HDDCT pulse with 100 mg BID of MB had some efficacy against Babesia and Bartonella but was insufficient to put most patients with active co-infections in full remission. “Among 11 patients who were Babesia FISH positive, three (27%) remained in remission, six improved (55%), and two (18%) had no change in sweats, chills, flushing, or dyspnea. “Among 19 patients (76%) with a history of Bartonella exposure, 15/19 improved (79%), and four patients (21%) had no change in symptoms” [The three-drug combination of a tetracycline, rifampin, and dapsone (100 mg BID), i.e., DDDCT, was shown to have clinical efficacy in the treatment of CLD/PTLDS, demonstrating tick-borne symptom improvements in 98% of patients, with 45% remaining in remission for 1 year or longer if they didn’t have active rtonella . In thatemission . Clinicaymptoms” .Bartonella. Research in the past several years has shown that Bartonella, such as Borrelia, can form persisters in biofilms [Bartonella-mediated endocarditis, representing a potential reservoir for the persistence of these bacteria [B. henselae to identify more effective treatments for persistent Bartonella infections [Bartonella henselae in vitro [Bartonella, with no viable bacteria detected after 6-day drug exposure [Bartonella, one of the primary co-infections interfering with the success of DDDCT and HDDCT [Bartonella persisters (the Hopkins study did not identify medication dosing) but would also have the advantage of helping to lower methemoglobin levels resulting from the use of higher dose dapsone [Bartonella who took higher doses of MB (300 mg BID) during their pulse therapies vs. lower doses (100–200 mg) noticed improved clinical results. Patient 23 noted sustained improvements in energy, muscle and joint pain, neuropathy, and muscle strength  with a higher dose of MB. Patient 24 similarly noticed for the first time sustained improvement in energy, myalgias, neuropathy, and cognitive functioning after using a 6-day HDDCT pulse with MB 300 mg PO BID. Patient 10 became 2 months symptom-free for the first time after a 4 day HDDCT pulse and 300 mg PO BID of MB, whereas lower doses of MB with 4 day pulses  helped decrease symptoms of fatigue, pain, neuropathy, brain fog, and Bartonella “pseudo seizures”  for a maximum of one month before relapsing during her menstrual cycle. She is now in full remission several months after doing her first 6-day HDDCT pulse with 300 mg PO BID of MB.Our present protocol was the first dapsone combination therapy to regularly use a 4-drug combination of dapsone with a tetracycline (minocycline or doxycycline), rifampin , and azithromycin. A 5th antibiotic, pyrazinamide, i.e., PZA (or rarely macrodantin if there was an intolerance to PZA), was also added in resistant cases, using longer courses of high-dose dapsone (5–7 days vs. 4-day regimens) with higher doses of methylene blue (up to 300 mg PO BID) for the treatment of CLD/PTLDS and associated co-infections, including biofilms ,239, andbacteria . Johns Hfections  and evalin vitro . Their cexposure . We, thend HDDCT ,103. Usi dapsone ,172, whi dapsone . SeveralBartonella in the majority of our patients based on the drug’s efficacy in mycobacterium infections, another bacterial “persister.” PZA has been published to help shorten the course of mycobacterium therapy by demonstrating efficacy in latent infections, phenotypic resistance, or tolerance [Bartonella published several years ago in a patient with Behçet’s disease, a severe autoimmune illness, demonstrated that dapsone combination therapy along with PZA had efficacy against a stealth infection with Bartonella, helping to relieve resistant symptoms [Bartonella. Among those patients who had CLD and Bartonella and who took 8 weeks of DDDCT and a 5–7 day course of HDDCT with PZA, 7/23 patients (30.5%) remained in full remission for 3 months or longer, and 3/23 (13%) were in remission for up to 6 weeks. Due to the small sample size and lack of a similar comparison group, we cannot conclude whether PZA increased response rates to Bartonella. Among three patients in our cohort with a history of CLD  without evidence of Bartonella who did not use PZA, 100% (3/3) went into long-term remission with an 8-week course of DDDCT and one 4-day HDDCT pulse. The 4-day HDDCT pulse was only effective in inducing long-term remission in those patients without evidence of active Babesia or Bartonella. This included patient 5, who met the criteria for PTLDS after having had an EM rash 21 years prior. He had low-grade symptoms for several years after doing an 8-week course of DDDCT. He improved significantly; however, symptoms would come and go. Relapses included several 10–14 day cycles per year, with a return of joint pain, irritability, insomnia, and word recall issues that would then spontaneously resolve. It was not until doing a 4-day HDDCT pulse with 300 mg PO BID of MB three years after taking one course of DDDCT that he went into full remission for over 6 months’ time (see Protocol 2). A double-blind, controlled, randomized trial using DDDCT followed by a 4-day HDDCT pulse needs to be conducted to confirm whether this protocol will lead to long-term remission in those patients with CLD without associated co-infections, as it did in patients 5, 6, and 7.We added Pyrazinamide (PZA) for the treatment of resistant olerance . A case symptoms . It was Bartonella  with striae and 3 species of Babesia  and had been sick for over 10 years before doing dapsone combination therapy. He had an EM rash in 2008 (meeting the criteria for PTLDS) and had previously failed 7 years of antibiotics before seeing us, including IV doxycycline for two months and IV azithromycin, as well as a short course of DSF. No antibiotic protocols during the 7 years of treatment had resulted in any significant sustained improvement. He did one course of DDDCT and two seven-day courses of HDDCT and had a 15% improvement in symptoms, including decreased muscle/joint pain, neuropathy, tinnitus, and improvements in cognition. Rotations of Babesia medications  along with dapsone were required to help decrease his sweats and air hunger, classical symptoms of Babesiosis. Dapsone’s benefit in this patient was in part from its antimalarial effects, i.e., antimicrobial as well as antiprotozoal effects, making it useful as an adjunct treatment of Babesia [Bb [Regarding potential side effects of the medication regimen, one patient, patient 23, was unable to take PZA due to a transient rash that developed on the medication. It was promptly resolved using H1 and H2 blockers  . He had  Babesia ,102,104; Babesia , with ef Babesia ,245; as esia [Bb  with excesia [Bb .Bartonella and multiple species of Babesia, like patient 23, were the sickest individuals in this cohort with the most severe, resistant symptoms. This may be due to the overlapping effects of Babesia and Bartonella each increasing inflammatory cytokine production [Borrelia and Babesia increasing inflammation, where each pathogen affects the other microbe directly or indirectly by influencing the host immune response [Bartonella species and Babesia species in CLD, where patients also had other overlapping sources of inflammation with downstream effects (MSIDS variables). There are presently over 36 species of Bartonella that have been identified, of which at least 17 have been associated with an expanding spectrum of animal and human diseases [Bartonella henselae (associated with a virulent form of cat scratch fever and culture-negative endocarditis), Bartonella quintana , Bartonella elizabethae, Bartonella vinsonii subspp., and Bartonella koehlerae  [Bartonella bacilliformis . Clinical manifestations of these Bartonella species include a broad range of eye problems , cardiac problems , peripheral neuropathy, central nervous system neurological disorders (Pediatric acute-onset neuropsychiatric syndrome (PANS), hallucinations, psychosis, encephalopathy, transverse myelitis, radiculitis, cerebellar ataxia), [Bartonella species have also been associated with breast cancer [Patients with multiple species of oduction ,246, as response . To the diseases ,36. We ftations) ,249, andataxia),  rheumatoataxia), , chronicataxia), . Recentlt cancer ,253,254 t cancer , a highlBartonella can overlap those of CLD, including fevers, fatigue, joint and muscle pain, headaches, cognitive difficulties, eye problems, and mood disorders [Bartonella may be present include severe neuropsychiatric symptoms with striae [Bartonella is present. We believe that Bartonella infections in our study interfered with the long-term efficacy of HDDCT, as did multiple Babesia species, necessitating a differential diagnosis and laboratory testing for multiple species of each infectious agent. Several of these Bartonella species have been associated with immunosuppression. These include B. bacilliformis, B. henselae, and B. quintana [Bartonella had Chronic Variable Immune Deficiency (CVID), i.e., patients 1, 8, 11, 22, and 24, and patient 23, who had 4 species of Bartonella , had Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). Considering the high incidence of immunosuppression in 5/25 patients (20%) as well as neuropathy  with neuropsychiatric symptoms among this cohort of patients, any patients presenting with CLD and associated co-infections with the above clinical symptomatology warrant initial laboratory testing for Bartonella species and immune deficiency. This evaluation should include an immunoglobulin panel  with IgG subclasses [Bartonella species is reasonable in this clinical setting, as well as other co-infections and MSIDS variables, since not only has Bartonella been associated with immune deficiency [Anaplasma [Babesia [Bartonella, which can lack adequate sensitivity [Bartonella spp. [Bartonella immunoblots [Bartonella FISH testing [Bartonella species. We found Bartonella FISH testing (IgeneX laboratories) to be positive in 11/25 patients (44%) and helpful in proving an active Bartonella infection as a cause of persistent symptoms when other serological methods were negative. The vascular endothelial growth factor (VEGF) test was also useful as an indirect marker of active Bartonella [The symptoms of isorders , potentih striae   have been shown to induce VEGF-mediated vasoproliferative disease in immunocompromised or immunocompetent animals  and humans [B. vinsonii subsp. berkhoffii, B. henselae, and other Bartonella spp. have developed survival mechanisms that allow for low-level intravascular and intracellular persistence [Babesia microti (1:320+), and Bartonella  with a history of severe fatigue, joint pain, sweats, and shaking chills (Babesia) with severe memory/concentration problems, depression, and panic attacks. She had been on high-dose morphine sulfate years ago before seeing us due to her severe pain, which was still not controlled by narcotics. She did two courses of DDDCT in the past several years, which improved her health and allowed her to stop all her narcotics as her pain levels decreased. She recently did one 6–7 day course of HDDCT with 300 mg of MB PO BID at the end of December 2022, several years later. After having a severe Herxheimer reaction, she improved from 25% to 87% of normal functioning, and a Bartonella granuloma on her hand decreased in size by approximately 80% . She did one 8-week course of DDDCT in August 2022, followed by a 6-day HDDCT pulse in March 2023, and had significant improvements in arthritic joint pain, energy, sleep, and cognition. She started at 23–30% of normal and was now functioning at 80% of normal as of May 2023. Finally, patient number 20 had a history of CLD, Babesia microti (1:64+), an indeterminate Bartonella immunoblot with Bartonella henselae (1:256+), and elevated VEGF. His primary symptoms in 2017 included severe fatigue, myalgias, rib pain, headaches, neuropathy, lightheadedness, cognitive difficulties, and mood swings. He did one 8-week course of DDDCT in March 2019 with doxycycline, rifampin, azithromycin, and pyrazinamide, and although he improved, one year later, in April 2020, symptoms relapsed after being treated for cellulitis with Augmentin when Bartonella striae emerged  and went from 50–60% normal to 95% normal functioning. He would have been counted as in remission if not for his occasional word-finding problems and sleep issues. This patient had multiple overlapping MSIDS variables affecting his resistant symptoms.fections . At leasd humans , as B. vsistence . We foun 80% see . She felrged see . The DDDBartonella in CLD, which is controversial, and whether it is definitely a tick-transmitted pathogen. Bartonella spp. can be transmitted from mammals to humans via cat fleas, sand flies, keds, mites, and human body lice [Bartonella via ticks, red ants, and spiders [Bartonella transmission by ticks, although one study in Europe with Ixodes ricinus ticks confirmed their ability to transmit a species of Bartonella, B. birtlesii [Bartonella from ticks, which have been shown to contain different Bartonella spp. [Bartonella [Bartonella henselae [Bartonella species. These included: 11 patients with Bartonella henselae; 7 with Bartonella quintana; 6 with Bartonella elizabethae; 6 with Bartonella vinsonii subspp.; 1 with Bartonella koehlerae; 1 with Bartonella bacciliformis; and 4 with Bartonella spp. (genus) without specific spp. identification. The MyLymeData patient registry of about 4000 participants highlighted the large heterogeneity of treatment responses for those with CLD [Bartonella and Babesia, undiagnosed/untreated MSIDS factors, as well as the large variability in treatments, including newer persister drug regimens, accounted in part for the large diversity of treatment outcomes.There are several unanswered questions regarding the etiology and role of ody lice . Recent  spiders . Furtherirtlesii . Whetherlla spp. ,274,275 rtonella ,102,103.henselae . In our with CLD . Based owith CLD ,105, it Babesia, a malarial-type parasite found in ticks [Babesia microti; 10 had Babesia duncani; 2 had Babesia odocoilei (FISH); 1 had Babesia divergens; and 20% (5/25) had evidence of active Babesia, i.e., were Babesia FISH positive. The primary symptoms present in those with active Babesia included day sweats, night sweats, chills, flushing, an unexplained cough, and “air hunger”, which are questions 1 and 22 on the HMQ-validated Lyme questionnaire [Babesia were more resistant to treatment, and none of the 6 patients (24%) who were Babesia FISH+ remained in full remission. Although the range of a B. microti lineage has been known to be expanding in the Northeastern US [the Southeastern US [B. divergens and Babesia venatorum [B. microti and B. duncani. Babesia duncani has also been increasing its range from the Western US to the Eastern US [Ixodes ticks is B. odocoilei [B. odocoilei has been found in ticks from Canada to Texas [B. odocoilei were found by Scott et al., implying that the parasite can be pathogenic in humans [B. odocoilei serologically cross-reacts with Babesia duncani [eight most prevalent Babesia species reported in humans from different parts of the globe include B. bigemina, B. bovis, B. crassa-like, B. divergens, B. duncani, B. microti, B. odocoilei, and B. venatorum [Babesia spp. have the highest prevalence in Europe, lower middle-income countries, and among individuals with a history of tick bites and other tick-borne diseases [in ticks , which cin ticks , maternain ticks ,280, andin ticks , was fouionnaire . Those pstern US  as well stern US , it has enatorum . Patientstern US ,285 as wstern US , and shodocoilei ,288,289.to Texas , and recn humans . Since B duncani , it is penatorum . These Bdiseases .Babesia oftentimes required multiple rotations of antimalarial therapies, including clindamycin [cryptolepis sanguinolenta, Scutellaria baicalensis (Chinese skullcap), Polygonum cuspidatum (Japanese knotweed) [B. microti, which has been implicated in a few cases of human babesiosis, all of them found in Europe [B. microti [Patients with active ndamycin , atovaqundamycin , atovaqundamycin , tafenoqndamycin , with orndamycin , lumefanndamycin , and botnotweed) , as wellnotweed) . Oftentinotweed) ,296; hownotweed) , and reqnotweed) . Clofazin Europe ,300. The microti . Many of microti ,302,303.Babesia microti has become resistant to standard therapies  [B. burgdorferi infection depends on a Th2 CD4+ T cell response within regional lymph nodes, and co-infection with B. microti may influence T cells toward a TH1 response [Babesia spp. may also complicate the course of DDDCT and HDDCT, as clinically they can result in hemolytic anemia [Babesia infections [medications’ side effects includes elevated levels of methemoglobin [Babesia  may also cause hemolytic anemia, it is incumbent on the physician to determine whether one or several etiologies are present. Although mild hemolytic anemia is common following dapsone treatment, it rarely warrants a change of therapy, as increased hemolysis usually only occurs in patients with glucose-6-phosphate dehydrogenase deficiency [quinine) . As per quinine) , the patresponse . Babesiac anemia ,307, simc anemia  used to fections ,294. Tafmoglobin , similarmoglobin , and Babficiency ,311. Meaficiency , or if tficiency . In the Bartonella to further improve tolerability, as well as in other autoimmune dermatological diseases such as dermatitis herpetiformis, where dapsone is used in doses up to 400 mg per day [Laboratory values were closely monitored during the first month on DDDCT (week 3), then weekly (weeks 5–8) during the second month on DDDCT, and biweekly during the last week of an HDDCT pulse. Despite changes in a number of laboratory values during the course of treatment, all patient values returned to normal at the end of treatment . Laboratory values with a CBC, CMP , and methemoglobin levels were monitored before, during, and after DDDCT and HDDCT. There were temporary changes in white cell counts , platelets (3 patients had mild thrombocytopenia), transient elevation in myelocytes and metamyelocytes , regular decreases in hemoglobin (Hb) and hematocrit (Hct), and expected rises in the mean corpuscular volume (MCV) while on dapsone due to the drug’s effect as a folic acid inhibitor, affecting bone marrow production . There w per day ,314,315. per day ,317.Liver functions were closely monitored on rifampin and PZA due to the potential of both medications to cause transaminitis ,319. ASTBartonella, when the protocols were adhered to, including the laboratory testing schedule. Higher doses of methylene blue and anti-oxidants, as well as folic acid supplementation, used in this HDDCT protocol compared to prior published higher dose dapsone protocols [We regularly used liver support with NAC, ALA, glutathione, and sulforaphane , and addrotocols ,103, mayBartonella. We found that 8 weeks of DDDCT followed by a 4-day pulse of HDDCT was sufficient to put patients with CLD/PTLDS into long-term remission if they did not have evidence of active co-infections, i.e., Babesia and Bartonella. Patients who were co-infected with Bartonella were sicker, with more neuropsychiatric symptoms, immune deficiency, and increased autonomic and peripheral neuropathy. They required 8 weeks of DDDCT followed by a 6–7-day pulse of HDDDCT to improve and/or go into full remission. The 4-day HDDCT, although helpful in relieving symptoms in those with CLD and Bartonella, was insufficient to keep most co-infected patients with Bartonella in remission.This is, to our knowledge, the first effective, short-term oral, generic protocol for treating CLD/PTLDS and associated co-infections, including Bartonella had superior results using 8 weeks of DDDCT followed by a 6–7-day pulse of HDDCT. This confirms the culture results from Johns Hopkins researchers looking at Bartonella persisters in culture, where 6 days of combination antibiotic therapy  were required to completely eliminate Bartonella persisters in biofilms [Bartonella. Pyrazinamide may also have been helpful in inducing remission rates for Bartonella; however, larger controlled studies will be necessary to determine its beneficial effect [Babesia and/or Bartonella. Three patients (13%) also had a full resolution of their Lyme and tick-borne symptoms for 1 ½ months or longer, having recently completed the full protocol 6 weeks prior, and age, length of illness, or gender did not affect remission rates or clinical improvement. Sixteen patients (64%) were sick for 10–20 years or longer, and 8/16 (50%) were in remission, with 8/16 (50%) showing significant clinical improvement.Patients with evidence of chronic biofilms . We did biofilms ,104,105.l effect ,326. Of We recognize that the tick-borne protocol presented here is complex, has potential gastrointestinal issues (nausea and vomiting) and hematological side effects with HDDCT, and requires a highly motivated health provider and a highly engaged patient. It is, however, a protocol that we have demonstrated to be safe and effective when patients follow the guidelines outlined in Bartonella) [Borrelia, Babesia, and Bartonella), dysbiosis, leaky gut with food sensitivities or mast cell activation syndrome, environmental toxins , mineral deficiencies, and chronic insomnia. These six factors contributed to chronic inflammation, resulting in downstream effects including mitochondrial dysfunction, POTS/dysautonomia, liver dysfunction, autoimmunity, and hormonal dysregulation. Despite these overlapping variables, 100% of our patients had improvement in their tick-borne symptoms using this short-term, oral, generic antibiotic protocol, implying a major role for the biofilm/persister forms of the bacteria, i.e., Borrelia and Bartonella, in driving chronic symptomatology.Climate change and the rise in global temperatures are one of the greatest threats to mankind’s health and are increasing our risk of vector-borne illness, whether it be mosquito-borne  , flea-botonella)  or tick-tonella) . Lyme ditonella) , and clitonella) . Borrelitonella) , fibromytonella) , neuropstonella) ,66,67. Ttonella) ,332, sectonella) . OverlapBabesia spp. that played a major role in interfering with the success of DDDCT and HDDCT, and if questions 1 and 22 on the HMQ Lyme questionnaire are positive [Babesia FISH-positive remained in long-term remission. It is essential that we invest more research funding into finding effective treatments for chronic babesia. Patients who are immunosuppressed, splenectomized, and/or elderly with chronic health conditions, including cardiac and pulmonary problems, are at greater risk for lethal complications from Babesia [Babesia microti is now resistant to standard anti-malarial therapies, with new species on the horizon, we need new tools and arsenals to fight these chronic parasitic infections, which are making chronic LD patients sicker. Investing in regular surveillance of ticks is also essential to determine the constantly changing environment of bacteria, viruses, and parasites [Babesia odocoilei being reported in the United States, and we lack sufficient knowledge regarding its spread among ticks in the Northern Hemisphere, the parasite’s pathogenicity, and/or how new emerging Babesia species may interact with the same host, causing illness. Similarly, several of our patients had simultaneous active co-infections with Lyme disease, different Babesia species, and different Bartonella species. We lack sufficient knowledge about the complex interactions of these organisms, and we suggest more research funding should also be invested in these areas.It was ultimately chronic positive , it shou Babesia . Since Barasites . Our papBartonella exacerbating psychiatric symptoms. If the patient presents with classic Bartonella striae , Bartonella FISH, and a Bartonella Immunoblot (IgeneX Laboratories) and/or T Labs . VEGF is also a useful surrogate marker for active Bartonella and can be obtained through local laboratories.Based on our patient population, anyone suffering from severe neuropsychiatric symptoms who also has significant physical complaints seen in CLD/PTLDS, i.e., chronic fatigue, headaches, migratory muscle/joint/nerve pain, cognitive difficulties, and a sleep disorder, should alert the clinician to the possibility of riae see  with or riae see , pain onBorrelia and Bartonella conducted at major universities [Bartonella.If a patient with CLD/PTLDS has failed multiple antibiotic protocols and has not yet tried a biofilm/persister protocol such as dapsone combination therapy, it is the first therapy that we would recommend based on our clinical experience, as long as there are no clear contraindications, i.e., G6PD deficiency, iron deficiency anemia, severe sulfa allergy, etc. Although the role of biofilm and persister bacteria in Borreliosis and Bartonellosis has been debated in the scientific literature, multiple publications on dapsone combination therapy have shown significant efficacy rates with short-term antibiotic therapies in the treatment of CLD/PTLDS using combination therapies with a persister drug, dapsone, along with biofilm agents. The chronic Lyme community has been desperate to find a cure for this chronic illness . The “beersities ,336,337 Babesia and Bartonella, biofilm/persister medications, as well as MSIDS factors, were not regularly accounted for in prior randomized controlled trials for CLD/PTLDS evaluating the efficacy of chronic antibiotic therapy [Multiple infections, including  therapy ,341,342. therapy . A preci


This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of using urine samples, which contain exfoliated tumor cells, to generate urine-derived BC organoids (uBCOs). Urine samples from 29 BC patients were collected and cells were isolated and cultured in a three-dimensional matrix. The establishment and primary expansion of uBCOs were successful in 83% of the specimens investigated. The culturing efficiency of uBCOs was comparable to cancer tissue-derived organoids. Immunohistochemistry and immunofluorescence to characterize the uBCOs exhibited similar expressions of BC markers compared to the parental tumor. These findings suggest that urine-derived BC organoids hold promise as a non-invasive tool for studying BC and evaluating therapeutic responses. This approach could potentially minimize the need for invasive procedures and provide a platform for personalized drug screening. Further research in this area may lead to improved diagnostic and treatment strategies for BC patients. With more than 570,000 newly diagnosed cases worldwide, bladder cancer (BC) accounts for one of the most common cancers in men and women in general . PrimaryIn recent times, three-dimensional bladder cancer organoids (BCO), derived from tumorous bladder cancer tissue (tBCO), were introduced into uro-oncological research. Several years ago, organoids were defined as three-dimensional in vitro cell culture constructs which contain cells colonizing on a porous matrix and hold the capability to generate tumor-like structures ,9,10. InBCOs hold the potential to facilitate drug development and lay the basis for an individualized multi-drug screening tool, as the histological and molecular spectrum of the maternal tumor is also represented within its derived organoids ,13,14. IOne of the major disadvantages of the establishment of patient-derived BCOs is displayed in the need for an invasive procedure  to harvest the malignant cells from the bladder to sow the organoids. Since BC exfoliates tumor cells into the patient’s urine, which is commonly used as a diagnostic tool in urine cytology, fluorescence in situ hybridization, nuclear matrix protein-22, and many others should also hold the potential to generate urine-derived bladder cancer organoids (uBCO) to spare the invasive procedure and thereby minimize the risk for the patient ,22,23. RIn this study, we evaluated the feasibility of establishing urine-derived tumor organoids directly from rinsing urine samples of BC patients without any intermediate host and compared the efficiency to tBCOs. We were able to establish a long-living urine-derived tumor line in a three-dimensional scaffold and found a comparable culturing efficiency for urine-derived and tissue-derived organoids. In addition, urine-derived cancer organoids show similar expression levels of antigens typical for BC, as well as tumor stem-cell markers when compared to their maternal tumor. Rinsing urine collected prior to TURBT was buffered immediately after collection with an equal volume of cold PBS  and refrigerated at 4 °C on wet ice until processing A. Urine 3, Assistant Germany, Sonderheim, Germany; Mikroskop Axiovert 135, Zeiss Germany, Jena, Germany), and the cell count of the sample was adjusted to 2 × 106 cells/mL by adding media or by further centrifugation and resuspension in less media. Then, cells were mixed on wet ice with three times the volume of base membrane extract , resuspended carefully, and plated out on 2–7 wells of a 48-well plate  as described recently (Urine samples were transferred to sterile 50 mL centrifugation tubes and centrifuged at 250 g for 10 min at room temperature (RT) . The urine supernatant was removed to 1 mL and the tissue pellet was resuspended, transferred to a tube, and washed with washing buffer C, the perecently D [25]. I2 for 15 min, and then each well was covered with 250 µL of bladder tumor medium ((BTM): 22.5 mL L-WRN conditioned medium, in-house product; 22 mL Advanced DMEM(1×)/F-12, Thermo Fischer, Waltham, MA, USA; 2.5 mL of 5% FBS, Sigma Aldrich, Taufkirchen, Germany; 1 mL B27 supplement, Gibco Life Technologies, Carlsbad, CA, USA; 500 μL L-glutamine, Biochrom, Berlin, Germany; 500 μL HEPES 1 M, Gibco Life Technologies, Carlsbad, CA, USA; 500 μL nicotinamide 1 M, Sigma Aldrich, Taufkirchen, Germany; 125 μL N-acetylcycsteine 500 mM, Sigma Aldrich, Taufkirchen, Germany; 50 μL A83-01 5 mM Tocris Bioscience, Bristol, UK; 50 μL Primocin 50 mg/mL, Thermo Fischer, Waltham, MA, USA; 50 μL FGF-10 100 μg/mL, Peprotech GmbH, Cranbury, NJ, USA; 25 μL FGF-7 50 μg/mL, Peprotech GmbH, Cranbury, NJ, USA; 12.5 μL FGF-2 50 μg/mL, Peprotech GmbH, Cranbury, NJ, USA; 5 μL Y-27632 100 mM, Hycultec, Beutelsbach, Germany, and 0.5 μL EGF 500 μM, Peprotech GmbH, Cranbury, NJ, USA). Representative domes were captured as light microscopy images and the cell culture was then incubated at 37 °C and 5% CO2. The 48-well plate was inverted and incubated at 37 °C and 5% COThe cultured bladder tumor cells were evaluated by light microscopy  at intervals of 1–3 days and assessed regarding their size increase and the morphology of the cultured cells/cell conglomerates E.The urine and tumor cell cultures were passaged when they showed strong growth of organoids and the supply of BTM, or when the integrity of the cast dome appeared to be at risk. Cells were also passaged when light microscopy showed the secretion of membrane vesicles as a sign of impending apoptosis. The splitting ratio was determined based on growth behavior and the estimated number of cells in the wells .6/mL to obtain 4 × 104 cells in 10 μL. Next, these cells were mixed with 30 μL basal membrane extract  on ice, transferred to a 24-well plate , and inverted to create hanging drops. After a short incubation at 37 °C, the plates were flipped back and supplemented with 500 μL of BTM culture medium per well. The plates were then incubated in a cell-culture incubator . All organoid cultures included in the study were downscaled to fit 48-well plates  [The culturing of tBCOs was performed according to the previously published protocol . In summME, USA) .To characterize the tumor organoids, two distinct methods of immunohistochemistry of paraffin sections and immunofluorescence were applied.Immunohistochemistry was utilized to detect the expression of the GATA-3, CK5/6, CK7, p53, p63, CD24, and CD44. The primary antibodies were washed off , and detected by the HRP or AP polymer reagent (IHC ZytochemPlus). The samples were counterstained by HE, covered , and recorded by microscopy  as described .For three-dimensional evaluation of the uBCOs, organoids were cultured in 8-well chamber slides. Immediately prior to staining, 100 µL dispase  was added to the medium. After incubation, the uBCOs were centrifuged and the supernatant was discarded. The uBCOs were fixed by 4% formaldehyde , washed well three times with PBS, blocked , and incubated  with primary antibodies to GATA-3, AE1/AE3, CK5/6, CK7, CK20, p53, TP63, Vimentin, s100P, CD24, CD44, Ki67, and FGFR3 at optimized concentrations (Tbl.S.1). Unbound primary antibodies were washed away . Primary antibodies were detected by incubation of the samples with complementary fluorescence-labeled secondary antibodies  as requested by the supplier. Unbound secondary antibodies were rinsed away . Cellular nuclei were counterstained by DAPI and the expression of the marker genes was visualized by microscopy (Leica Stellaris 8 or Zeiss Axiophot). The antibody diluent was 1% BSA in PBS. Samples omitting the primary antibodies and samples stained with mouse or rabbit IgG isotype antibodies served as controls .p values < 0.05 were considered statistically significant. The workflow was created using biorender.com (accessed 12 May 2023).Data are displayed as median if applicable and box plots are displayed as median with 25th or 75th quantiles and min/max whiskers. Individual groups were tested using the Mann–Whitney U-test. Spearman’s rho (ρ) was calculated for correlation between continuous data. A two-sided Fisher exact test was used to evaluate the differences in efficiency rates. Graphs were plotted using GraphPad Prism v.8.4.0. Statistical analyses were conducted using GraphPad Prism v.9.4.1 and JMP  software. Urine samples from a total of 29 patients with pathologically confirmed, active urothelial cancer at primary diagnosis or relapse were included in this study. In 13/29 patients (45%), concomitant tumor tissue was collected for the establishment of additional tBCOs. The median age of the patients at the sample collection was 66 years (range: 30–85). Among the patients, 83% were male. The majority of patients (69%) had NMIBC and 59% showed high-grade histopathological features. A total of 72% of patients had positive urine cytology at the time of sample collection. Tumor-specific treatment was applied in 45% of patients prior to sample collection, with local surgery (34%) or instillation (7%) therapies applied most commonly. Detailed patient characteristics are shown in For the establishment of uBCOs cells from a median of 280 mL (100–400 mL), rinsing urine was cultured in a three-dimensional matrix after centrifugation and washing .p = 0.55; p = 0.06; p = 0.06; p = 0.21; p = 0.36; p = 0.28; Cultures of uBCOs were successfully established for 83% of specimens in the first passage. The subsequent expansion of uBCOs was achieved for 62% of samples, with 7% reaching the fifth passage A. The cup = 1.00), 62% and 46% (p = 0.50), 37% and 8% (p = 0.06), 28% and 7% (p = 0.23), and 7% and 0% (p = 1.00) for passages 1 to 5, respectively  was performed using immunohistochemistry and multidimensional immunofluorescence. The uBCOs showed, in general, positivity for markers of urothelial differentiation , cytokeratins typically expressed in BC , and for the proliferation marker Ki67. TP63 and p53 showed positivity in 67% and 100% uBCOs, respectively. A total of 50% of uBCO showed mesenchymal differentiation with positivity for vimentin. Immune checkpoint antigen CD276, also known as B7-H3, and stemness markers, the sialoglycoprotein CD24, and cell-surface glycoprotein CD44 showed positivity in 67%, 33%, and 67%, respectively  and in cases of low-grade carcinomas (p = 0.06). However, it is important to reevaluate this trend in a larger group of patients to obtain more conclusive results.The culturing efficiency of uBCO was found to be similar to the rates of successful organoid formations in a canine urinary-derived BCO model . CompareLight microscopy revealed a comparable morphology and three-dimensional architecture of uBCOs and tBCOs, as well as reported appearances of BCOs ,27. The The establishment of uBCOs offers distinct advantages over tBCOs. In this feasibility trial, rinsing urine was utilized to simultaneously acquire comparable tissue samples alongside subsequent TURBT procedures. Going forward, the focus should shift to employing voiding urine samples to enhance patient comfort; reduce invasiveness; and, subsequently, lower potential risks. Furthermore, uBCOs could serve as a foundation for drug testing and the exploration of personalized therapeutic strategies, as they faithfully reflect the phenotypical attributes of the primary tumor. The approach of using voiding urine would also enable the creation of uBCOs in parallel with the treatment timeline, presenting a real-time ex vivo/in vitro screening tool. Further trials are imperative to investigate the feasibility of utilizing voiding urine instead of rinsing urine for uBCO generation.Although the results hold promise, it is important to acknowledge several limitations. The trial’s feasibility-oriented design did not allow for investigating specific cell populations, such as immune cells, in both the parental tumor and the generated uBCOs or tBCOs. Though the comparison between the parental tumor and its derived uBCOs could have been offered, the limitations imposed by the available materials hindered a simultaneous immunohistochemical or immunofluorescence characterization of both uBCOs and tBCOs in comparable, higher passages. Additionally, due to a relatively small cohort, it is imperative to validate these findings with a larger group of bladder cancer patients to ensure the reproducibility and broader applicability of the outcomes. Furthermore, though the evaluation of specific markers displayed consistency with the parental tumor tissue, a more comprehensive understanding of their similarity would necessitate further molecular characterization and a comparative analysis of genetic alterations between uBCOs, tBCOs, and primary bladder cancer tissue.Taken together, this study demonstrates the feasibility and comparable efficiency of establishing uBCOs from rinsing urine samples of BC patients. These uBCOs faithfully recapitulate the histological and molecular features of BC and offer a non-invasive approach for personalized tumor modeling and individualized drug screening. Further research and validation are warranted to fully explore the potential of urine-derived organoids in BC research and clinical applications.


Background: Previous studies have revealed the significant roles of SHC SH2 domain-binding protein 1 (SHCBP1) in occurrence and progression of cancers, but there is no pan-cancer analysis of SHCBP1.Methods: In this study, we explored the potential carcinogenic role of SHCBP1 across 33 tumors from the TCGA and GTEx databases. We investigated SHCBP1 expression, prognosis, genetic alterations, tumor mutational burden (TMB) score, microsatellite instability (MSI) and tumor microenvironment from TIMER2, GEPIA2, UALCAN and cBioPortal databases. Moreover, the cellular functions and potential mechanisms were evaluated by GO and KEGG analysis. Besides, the mRNA expression of SHCBP1 was examined using qRT-PCR assay in gastrointestinal cancers.Results: SHCBP1 was significantly upregulated in various cancers, and apparent relationship existed between SHCBP1 and survival prognosis in patients. The TMB, MSI, and tumor microenvironment analysis indicated that SHCBP1 was closely related to immune checkpoints, immune targets, as well as CD4+ naive T cell, CD8+ T cell, and neutrophil. Moreover, the cellular functions of SHCBP1 were mainly in regulating cell cycle motor protein activity. In addition, we validated that SHCBP1 mRNA expression was over-expressed in gastrointestinal cancers.Conclusions: This study was the first to systematically determine the prognostic value of SHCBP1, providing a forward-looking perspective on immunotherapy and cellular processes in pan-cancer. Cancer is the leading proportion of chronic disease cases, with an increasing incidence, posing a significant threat to public health worldwide , 2. MainSrc homolog and collagen (SHC) are a key adaptor protein of cell surface receptors . SHC SH2In this study, we explored and verified the potential roles of SHCBP1 in cancer development and progression via pan-cancer analysis, focusing on expression, prognosis, genetic alterations, protein phosphorylation, immune infiltration, and other relevant cellular processes. This comprehensive analysis highlights the multifaceted role of SHCBP1 and reveals its potential molecular mechanisms in generalized cancers.P≤0.01, log2|fold change| (FC) = 1.  [http://ualcan.path.uab.edu/analysis-prot.html) [We assessed SHCBP1 expression in tumors and normal tissues using the TIMER2 database with the following thresholds: nalysis) . The GEPnalysis) . For surot.html) .The overall survival (OS) and disease-free survival (DFS) in the GEPIA2 database were applied to evaluate the prognostic value of SHCBP1 in different cancers. The survival map was obtained using the threshold 95% confidence intervals. The first progression (FP), disease-specific survival, post-progression survival (PPS), progression-free survival (PFS), and relapse-free survival (RFS) correlations were assessed in Kaplan–Meier plotter to determine the prognostic value in breast, lung, ovarian, GC, and liver cancer with the threshold value as: “auto select best cutoff” and log-rank P values .We used the “TCGA PanCancer Atlas study” module to predict SHCBP1 alterations in the cBioPortal website with the following parameters: “Quick By Gene” and “SHCBP1” . The altSpearman correlation analyses were used to calculate the correlations of SHCBP1 expression in pan-cancers with tumor mutational burden (TMB) score, microsatellite instability (MSI), and two immune checkpoint pathway genes , with the results presented in bubble charts . To furthttps://string-db.org/) with low confidence of 0.150. Then, based on the TCGA and GTEx datasets, the first 100 gene correlations were extracted from the “Gene_Corr” module of TIMER2. The intersection map of SHCBP1 was plotted using the Venn viewer Jvenn. The gene intersections were employed to generate a heatmap. In addition, according to the two gene datasets, we performed GO and KEGG enrichment analyses with the R packages “ClusterProfiler” and “ggplot2.” Cnetplot was used to show the molecular functions [The “SHCBP1” and “Homo sapiens” parameters were used to acquire the top 50 SHCBP1-related altered genes using the STRING database  and human normal colonic epithelial cells (NCM460) were purchased from Procell Life Science . Human gastric cancer cells (7901 and AGS), human gastric mucosa epithelial cells (GES), HCC cells (HepG2 and Huh), and human liver cells (LO2) were obtained from the School of Basic Medicine, Anhui Medical University. Cells were all cultured in DMEM supplemented with high glucose (HyClone) and 10% fetal bovine serum  at 5% CO2 and 37° C.−ΔΔCt method. The primer sequences used for the experiment were as follows: 5′-GCTACCGTGATA AACCAGGTTC-3′ (forward) and 5′-AGGCTCTGAATCGCT CATAGA-3′ (reverse).The total RNA content of tissues and cells was extracted using TRIzol reagent (Takara). According to the instructions, a PrimeScript™ kit (Takara) was used to perform reverse transcription. SYBR Green qPCR Mix was used to detect the relative expression of target genes based on the 2P<0.05.Student’s t-tests or one-way ANOVA analyses were employed to determine the significance of differential SHCBP1 expression in two groups or multiple groups, respectively. The OS, RFS, DFS, and DMFS were analyzed using the hazard ratios (HRs) and P values  between high- and low-risk groups or altered and unaltered groups. Spearman’s analysis was used to investigate the relationship between SHCBP1 expression and immune infiltration. Statistical significance was defined as The datasets obtained and explored in this study are available in the TCGA, GEPIA2, UACLAN, cBioportal and TIMER2 databases.P<0.001) of SHCBP1 in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe carcinoma (KICH), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC). In addition, SHCBP1 expression was elevated (P<0.01) in cervical squamous cell carcinoma, endocervical adenocarcinoma (CESC), and pheochromocytoma (P<0.01). Finally, SHCBP1 expression was elevated (P<0.05) in paraganglioma (PCPG), kidney renal clear cell carcinoma (KIRC), and skin cutaneous melanoma (SKCM). In total, 21 types of cancers showed elevated expression of this protein (P<0.05) in adrenocortical carcinoma (ACC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), ovarian serous cystadenocarcinoma (OV), thymoma (THYM), and uterine carcinosarcoma (UCS) than in the corresponding normal tissues. However, the expression of SHCBP1 showed no significant difference in pancreatic adenocarcinoma (PAAD), acute myeloid leukemia (AML), lower-grade glioma (LGG), sarcoma (SARC), or testicular germ cell tumors (TGCT) . Low expression of SHCBP1 was also associated with longer DFS in PRAD and SARC but not with higher OS . In patients with PRAD, the unaltered group showed improved DFS (P=7.004e3) and PFS (P=0.049) but unchanged disease-specific survival  is highest in uterine corpus endometrial carcinoma (UCEC), in which “mutation” accounts for the primary proportion of alterations. PRAD showed the highest incidence of “amplification” alterations with a frequency of ~3% . We thenP=0.741, .We further estimated the role of SHCBP1 expression in modulating immune checkpoints. There were 24 immune inhibitors and 36 activators investigated across the various cancers. The expression of SHCBP1 was positively correlated with the expression of four inhibitors  and five activators  in the majority of tumors. However, in THYM, SHCBP1 expression was negatively correlated with the expression of all studied immune checkpoints except for KIR2DL3, EDNRB, ARG1, IL13, VTCN1, BTLA, IDO1, CD274, IL1O, TGFB1, IL1A, BTN3A1, IFNA2, PRF1, IL2, SELP, and TNFRSF14. CD276, VEGFA, TNFRSF9, TNFSF4, and HMGB1 play important immune roles in various tumors . Gene muThe mutation rate of tumors is closely associated with immune cell function , 27. TheTo further determine the role of SHCBP1, we employed enrichment analysis based on SHCBP1-binding proteins and co-expressed genes using the STRING and GEPIA2 databases. The analysis of the STRING database generated 50 SHCBP1-binding genes using data from previous experiments, which were plotted in interaction networks . Then, wTo validate the above results, we detected the expression of SHCBP1 in GC, CRC, and HCC cell lines. The experimental results indicated that SHCBP1 expression was upregulated in GC (AGS and 7901 cell lines), CRC (SW480 and HCT116 cell lines), HCC (HepG2 cell line) cells compared to their corresponding control groups –9C.in vitro previously, SHCBP1’s role in pan-cancer remains unclear.SHCBP1 involved in various physiological and pathological processes, such as cytokinesis during mitosis and meiosis . RecentlIn this study, we analyzed the expression of SHCBP1 in 33 tumor types, and SHCBP1 expression was elevated in most of these types except for PAAD, AML, LGG, SARC, and TGCT when compared with the corresponding normal tissues. Moreover, our experimental results verified that SHCBP1 expression was upregulated in the liver, gastric, and colorectal cancer cell lines, which was consistent with public database information. In addition, we found that SHCBP1 protein expression was also upregulated in BRCA, GBM, HNSC, LUAD, PAAD, and UCEC. High expression of SHCBP1 was correlated with tumor pathological stage in several cancers. In addition, the GEPIA database analysis showed that upregulated SHCBP1 was negatively correlated with patients' OS and DFS in ACC, KIRP, LGG, LIHC, LUAD, MESO, and PAAD. However, elevated SHCBP1 was correlated with a better prognosis in patients with GC. This is inconsistent with previous studies, which may be due to different analysis methods for different databases and studies. In general, SHCBP1 can promote tumor growth and invasion in GC by regulating the CDK4-cyclin D1 cascade and caspase-3 and caspase-PARP-dependent apoptosis pathways . Our resThe SHCBP1 gene is located in a region of chromosome 16q11.2 . In the Tumor immune escape is often due to the disruption of immune cell-mediated checkpoint pathways by tumor cells , 31. HowThis study had several limitations. Firstly, different database algorithms may cause errors in the analysis . SecondlThis study is the first to systemically explore the roles of SHCBP1 in pan-cancer. SHCBP1 expression was associated with clinical prognosis, genetic alterations, immune checkpoint expression, TMB, MSI, immune cell infiltration, and cellular processes, which provided a forward-looking view for effective tumor diagnosis and treatment. Future studies should further illuminate the potential mechanisms by which SHCBP1 modulates the immune microenvironment and lay a solid foundation for therapeutic target.Supplementary Figure 1