Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Bartonella henselae in a dog with ear tip vasculitis.

BACKGROUND: Bartonella henselae, a Gram-negative, zoonotic, alpha-proteobacteria has been previously implicated in association with cutaneous vasoproliferative lesions (bacillary angiomatosis), nodular panniculitis and multifocal erythema (erythema multiforme) in dogs. OBJECTIVE: Describe clinical, microbiological and histological lesions in a dog with ear margin vasculitis and B. henselae infection. ANIMALS: A 12-month-old, specific pathogen-free intact female beagle dog maintained in a vector-free laboratory animal resource facility. METHODS AND MATERIALS: Bartonella and Rickettsia serological evaluation, Bartonella and Rickettsia PCR, Bartonella alpha-proteobacteria growth medium (BAPGM) enrichment blood culture/PCR, histopathological investigation and confocal immunohistochemical evaluation. RESULTS: Serological investigation (seroreversion) and PCR testing of aural tissue biopsies failed to support Rickettsia rickettsii as a cause of the aural vasculitis; however, B. henselae, genotype San Antonio 2 DNA was amplified and sequenced from both ear tip margins and from normal-appearing abdominal skin. Seroconversion to B. henselae was documented retrospectively by IFA testing. Bartonella henselae organisms were visualized by confocal immunostaining within all three biopsies. Histopathology revealed small vessel necrotizing vasculitis and dermal necrosis. Bartonella henselae seroreversion and complete resolution of skin lesions occurred in conjunction with administration of oral doxycycline and enrofloxacin for six weeks. CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella henselae is an emerging zoonotic pathogen that has been associated with leucocytoclastic vasculitis in humans and may have had a contributing or causative role in the development of the cutaneous aural margin vasculitis in this beagle.

Mast cell activation contributes to sickle cell pathobiology and pain in mice.

Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.

Bartonella- and Borrelia-Related Disease Presenting as a Neurological Condition Revealing the Need for Better Diagnostics.

The diagnostic tests available to identify vector-borne pathogens have major limitations. Clinicians must consider an assortment of often diverse symptoms to decide what pathogen or pathogens to suspect and test for. Even then, there are limitations to the currently available indirect detection methods, such as serology, or direct detection methods such as molecular tests with or without culture enrichment. Bartonella spp., which are considered stealth pathogens, are particularly difficult to detect and diagnose. We present a case report of a patient who experienced a spider bite followed by myalgia, lymphadenopathy, and trouble sleeping. She did not test positive for Bartonella spp. through clinically available testing. Her symptoms progressed and she was told she needed a double hip replacement. Prior to the surgery, her blood was submitted for novel molecular testing, where Bartonella spp. was confirmed, and a spirochete was also detected. Additional testing using novel methods over a period of five years found Bartonella henselae and Borrelia burgdorferi in her blood. This patient's case is an example of why new diagnostic methods for vector-borne pathogens are urgently needed and why new knowledge of the variable manifestations of Bartonellosis need to be provided to the medical community to inform and heighten their index of suspicion.

Quercetin-3-methyl ether inhibits lapatinib-sensitive and -resistant breast cancer cell growth by inducing G(2)/M arrest and apoptosis.

Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin-3-methyl ether caused a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G(2)/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G(1) phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients.

Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids.

Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of mu opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.

Host tRNA-Derived RNAs Target the 3'Untranslated Region of SARS-CoV-2.

The COVID-19 pandemic revealed a need for new understanding of the mechanisms regulating host-pathogen interactions during viral infection. Transfer RNA-derived RNAs (tDRs), previously called transfer RNA fragments (tRFs), have recently emerged as potential regulators of viral pathogenesis. Many predictive studies using bioinformatic approaches have been conducted providing a repertoire of potential small RNA candidates for further analyses; however, few targets have been validated to directly bind to SARS-CoV-2 sequences. In this study, we used available data sets to identify host tDR expression altered in response to SARS-CoV-2 infection. RNA-interaction-prediction tools were used to identify sequences in the SARS-CoV-2 genome where tDRs could potentially bind. We then developed luciferase assays to confirm direct regulation through a predicted region of SARS-CoV-2 by tDRs. We found that two tDRs were downregulated in both clinical and in vitro cell culture studies of SARS-CoV-2 infection. Binding sites for these two tDRs were present in the 3' untranslated region (3'UTR) of the SARS-CoV-2 reference virus and both sites were altered in Variants of Concern (VOCs) that emerged later in the pandemic. These studies directly confirm the binding of human tDRs to a specific region of the 3'UTR of SARS-CoV-2 providing evidence for a novel mechanism for host-pathogen regulation.

The itchy scalp--scratching for an explanation.

Scalp pruritus is a common complaint that is considered a diagnostically and therapeutically challenging situation. Scalp skin has a unique neural structure that contains densely innervated hair follicles and dermal vasculature. In spite of the recent advances in our understanding of itch pathophysiology, scalp itching has not been studied as yet. In this review, we summarize the current knowledge on the neurobiology of scalp and hair follicles as well as itch mediators and provide a putative mechanism for scalp itch with special emphasis on neuroanatomy and pathophysiology.

Formaldehyde Release From Predispersed Tattoo Inks: Analysis Using the Chromotropic Acid Method.

BACKGROUND: Allergic contact dermatitis to tattoo ink may last from weeks to years. Formaldehyde is a strong sensitizer that may be present in predispersed tattoo inks. OBJECTIVES: The aim of this study was to evaluate the presence of formaldehyde in predispersed tattoo inks using the chromotropic acid method. METHODS: Tattoo inks from 39 companies were evaluated. Inclusion criteria included availability to purchase inks online through US tattoo product wholesalers or individual Web sites. Brands were grouped based on prevalence of use: common, uncommon, or rare. For common brands, 8 colors (primary colors, secondary colors, black, and white) were purchased. For uncommon and rare brands, 5 colors (primary colors, black, and white) were purchased. Each ink was tested with standard chromotropic acid method procedures; concentration of formaldehyde released was quantified using spectrophotometry. RESULTS: In total, 127 tattoo inks were purchased and tested. Ninety-three (73%) tested positive for formaldehyde release; 34 (27%) tested negative. Formaldehyde release did not correlate with color or brand. At least 1 ink from all brands (except 1) was positive for formaldehyde release. CONCLUSION: Approximately three-quarters of selected US tattoo inks tested positive for formaldehyde release. Clinicians should be aware of tattoo ink as a potential source of formaldehyde.

Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study.

Bartonella bacilliformis (B. bacilliformis), Bartonella henselae (B. henselae), and Bartonella quintana (B. quintana) are bacteria known to cause verruga peruana or bacillary angiomatosis, vascular endothelial growth factor (VEGF)-dependent cutaneous lesions in humans. Given the bacteria's association with the dermal niche and clinical suspicion of occult infection by a dermatologist, we determined if patients with melanoma had evidence of Bartonella spp. infection. Within a one-month period, eight patients previously diagnosed with melanoma volunteered to be tested for evidence of Bartonella spp. exposure/infection. Subsequently, confocal immunohistochemistry and PCR for Bartonella spp. were used to study melanoma tissues from two patients. Blood from seven of the eight patients was either seroreactive, PCR positive, or positive by both modalities for Bartonella spp. exposure. Subsequently, Bartonella organisms that co-localized with VEGFC immunoreactivity were visualized using multi-immunostaining confocal microscopy of thick skin sections from two patients. Using a co-culture model, B. henselae was observed to enter melanoma cell cytoplasm and resulted in increased vascular endothelial growth factor C (VEGFC) and interleukin 8 (IL-8) production. Findings from this small number of patients support the need for future investigations to determine the extent to which Bartonella spp. are a component of the melanoma pathobiome.

Effects of gefitinib (Iressa) on mammary cancers: preventive studies with varied dosages, combinations with vorozole or targretin, and biomarker changes.

The ability of the epidermal growth factor receptor inhibitor gefitinib (Iressa) to prevent/treat methylnitrosourea (MNU)-induced mammary cancers and to modulate biomarkers in female Sprague-Dawley rats was examined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. In the prevention studies, continual treatment with Iressa at 10, 3, or 1 mg/kg body weight per day beginning 5 days after MNU reduced tumor multiplicity by 93%, 43%, and 20%, respectively. Treatment of rats bearing small palpable cancers with Iressa (10 mg/kg body weight per day) resulted in the complete regression of 70% of the tumors. Short-term treatment of tumor-bearing rats with Iressa caused decreases in cell proliferation and phosphorylated epidermal growth factor receptor and increases in apoptosis. To examine treatment regimens that might decrease the skin toxicity associated with Iressa, both intermittent treatments and combinations of lower doses of Iressa with other effective agents were evaluated. Treatment with Iressa (10 mg/kg body weight per day) continually or intermittently (either "3 weeks on/3 weeks off" or "4 days on/3 days off") reduced cancer multiplicity by 91%, 24%, and 68%, respectively. However, all regimens reduced tumor weights >85%. Finally, combining suboptimal doses of Iressa with suboptimal doses of vorozole (an aromatase inhibitor) or targretin (a retinoid X receptor agonist) yielded greater chemopreventive efficacy than any of these agents given alone.

Formaldehyde Release From Baby Wipes: Analysis Using the Chromotropic Acid Method.

BACKGROUND: Formaldehyde is a common preservative and strong sensitizer. OBJECTIVE: The aim of the study was to evaluate the release of formaldehyde from baby/toddler wet wipes using the chromotropic acid method (CAM). METHODS: An online search of best-selling baby wipes was conducted. None declared formaldehyde or formaldehyde-releasing preservatives. Standard CAM procedures were used: a 1 × 1-in square of fresh wipe was placed in a bottle with an open vial of 4 mg/1 mL of chromotropic acid and sulfuric acid solution, sealed, and stored for 48 hours. Formalin and water served as controls. A blinded investigator graded color change (negative, indeterminate, mild, moderate, or strong). For quality control, 20% of all samples as well as all positives were retested. RESULTS: Fifty-one popular and highly reviewed baby and toddler wet wipe products were tested using CAM. Twelve wipes (24%) released formaldehyde (8 mild, 4 moderate/strong). Chromotropic acid method testing of 9 wipes (18%) was indeterminate and 30 (59%) were negative. CONCLUSIONS: Almost one quarter of baby/toddler wet wipes released formaldehyde when evaluated with CAM. Patients and clinicians should be aware of this potentially undeclared source of this common allergen.

Formaldehyde in "Nontoxic" Nail Polish.

BACKGROUND: Nail polish is known to contain potentially hazardous chemicals that have been linked to adverse health effects after overexposure. Formaldehyde is used as an antimicrobial, preservative, and nail hardener in select nail products, yet it is a recognized carcinogen and potent allergen in allergic contact dermatitis. OBJECTIVE: The aim of this study was to investigate whether formaldehyde is present in nail polishes marketed as formaldehyde-free. METHODS: Twenty-nine cosmetic nail polishes were purchased for analysis; of these, 28 were advertised as formaldehyde-free and/or did not declare formaldehyde in their ingredient lists. Initial testing was pursued using the chromotropic acid method, which uses a red-purple color change to indicate the presence of formaldehyde. Products were subsequently analyzed at least twice using high-performance liquid chromatography, quantifying formaldehyde amount above the detection limit of 2 ppm. CONCLUSIONS: High-performance liquid chromatography analysis found 5 of 29 products containing formaldehyde, 4 of which were advertised as formaldehyde-free. All other products were negative for formaldehyde (<2 ppm). Further investigation is warranted among brands testing positive and whether multiple products within the same line contain formaldehyde. Nail products must be labeled appropriately to avoid adverse reactions among individuals with cutaneous sensitivities.

Formaldehyde Release From Clothing and Upholstery Fabrics Using the Chromotropic Acid Method.

BACKGROUND: Formaldehyde resins may be used in textiles. OBJECTIVE: The aim of the study was to investigate the presence of formaldehyde in textiles using the chromotropic acid method. METHOD: Clothing scraps (from local department store tailors, n = 77) and upholstery fabric cuttings (from a furniture reupholstery store, n = 22) were collected. Each fabric was cut into a 1-cm square and tested using the chromotropic acid method. Samples were retested in a systematic fashion (every 10th sample) to assess reproducibility. RESULTS: All 99 clothing and upholstery fabrics tested negative for formaldehyde release. CONCLUSIONS: Our study suggests that textile manufactures may be using nonformaldehyde resins for durable press finishing in clothing likely to be tailored as well as fabrics used for furniture reupholstery. Additional studies involving other metropolitan areas and a variety of fabrics are needed to confirm these findings.

Bartonella henselae Infection in Sickle Cell Disease Mice Is Associated with Hyperalgesia.

BACKGROUND: Sickle cell disease (SCD) is the most prevalent hematologic genetic disorder. Acute vaso-occlusive painful crisis is the hallmark of the disease and may be related to subclinical infections. Bartonellosis, a rare and neglected infection, is caused by Bartonella spp., which can be found in donated blood. These bacteria cause intraerythrocytic and endothelial infection and pain, all of which occur in SCD. It is likely that this infection is transmitted to SCD patients during transfusion from donated blood, leading to pain. We, therefore, evaluated whether Bartonella henselae infection would cause hyperalgesia in mice with SCD. MATERIALS AND METHODS: SCD mice were generated by transplantation of nucleated bone marrow cells harvested from transgenic Berkeley sickle mice into 2-month-old irradiated C57BL/6 mice. We infected four SCD mice by intraperitoneal inoculation with B. henselae, and inoculated four other mice with the same volume of saline. Mechanical hyperalgesia was determined using von Frey monofilaments by two blinded observers. Thereafter, the animals were anesthetized and euthanized to collect blood, liver, and spleen samples to seek B. henselae infection by PCR. FINDINGS: We confirmed the experimental infection in all animals by PCR. Tremors and mechanical hypersensitivity were demonstrated by SCD mice infected with B. henselae infection but not in those receiving saline. CONCLUSION: B. henselae infection may be related to pain and other symptoms in SCD.

Local irradiation in combination with bevacizumab enhances radiation control of bone destruction and cancer-induced pain in a model of bone metastases.

Skeletal metastases are a major source of morbidity for cancer patients. The purpose of this study was to evaluate the effects of megavoltage irradiation and antiangiogenic therapy on metastatic bone cancer. A tumor xenograft model was prepared in C3H/Scid mice using 4T1 murine breast carcinoma cells. Twenty-eight mice bearing tumors were treated with either bevacizumab (15 mg/kg), local megavoltage irradiation (30 Gy in 1 fraction), combination of bevacizumab and local megavoltage irradiation or physiologic saline solution (control group). Tumor area, bone destruction, tumor microvessel density, pain-associated behaviors and expression of substance P were assessed. Combined modality treatment reduced the frequency of pain-associated behaviors, decreased levels of nociceptive protein expression in the spinal cord, maintained cortical integrity and decreased the density of microvessels as compared to single modality treatments. We conclude that concurrent antiangiogenic therapy and localized radiotherapy for the treatment of bone metastases warrants further evaluation in human clinical trials.

Phenotypic analysis of T-cells in extensive alopecia areata scalp suggests partial tolerance.

This study demonstrates the feasibility and efficacy of using flow cytometric analysis with intracellular cytokine staining for characterization of T-cell phenotype and functional status in extensive alopecia areata (EAA) scalp skin. Cell suspensions were made from scalp punch biopsies taken from 12 patients with long-standing EAA (average disease duration 14 years, 95% hair loss) and six control subjects. EAA samples had a lower percentage of CD-3-expressing cells, but CD-4/CD-8 ratios remained similar to controls. Expression of CD-69 was found only in EAA scalp biopsies, suggesting that T-cells from EAA scalp have undergone activation. No difference was found in tumor necrosis factor alpha expression. Surprisingly, EAA scalp T-cells produced less IL-2 and CD-8 T-cells produced less IFN-gamma. Immunohistochemical staining of formalin-fixed paraffin-embedded specimens demonstrated that IFN-gamma-producing cells in EAA scalp were not greater in number than in normal specimens. The few identified IFN-gamma-producing cells demonstrated no tendency to localize to the perifollicular region, and were similarly distributed as in control specimens. The abnormalities in cytokine production may explain the relative paucity of inflammatory change observed in the clinical setting and suggest that T-cell responses in EAA scalp are tightly, albeit aberrantly, regulated via mechanisms of peripheral T-cell tolerance.

Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance.

The skin as a barrier and immune organ is exposed to omnipresent environmental challenges such as irradiation or chemical and biologic hazards. Neuropeptides released from cutaneous nerves or skin and immune cells in response to noxious stimuli are mandatory for a fine-tuned regulation of cutaneous immune responses and tissue maintenance and repair. They initialize host immune responses, but are equally important for counter regulation of proinflammatory events. Interaction of the nervous and immune systems occurs both locally - at the level of neurogenic inflammation and immunocyte activation - and centrally - by controlling inflammatory pathways such as mononuclear activation or lymphocyte cytokine secretion. Consequently, a deregulated neurogenic immune control results in disease manifestation and frequently accompanies chronic development of cutaneous disorders. The current understanding, therapeutic options, and open questions of the role that neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide, neuropeptide Y, or others play in these events are discussed. Progress in this field will likely result in novel therapies for the management of diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm.