Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior.

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF neuronal subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three main BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamatergic neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under the control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed that cholinergic, GABAergic and vGluT2+ neurons represent distinct BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium-binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2-enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal or aversive/rewarding behavior such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2+ neurons elicited a striking avoidance of the area where stimulation was given, whereas stimulation of BF parvalbumin or cholinergic neurons did not. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest that BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.

Novel cannabidiol sunscreen protects keratinocytes and melanocytes against ultraviolet B radiation.

Cannabidiol (CBD), a natural occurring phytocannabinoid, is used extensively in consumer products ranging from foods to shampoos, topical oils and lotions. Several studies demonstrated the anti-inflammatory and antioxidative properties of cannabidiol. Nevertheless, the role of cannabidiol use in sunscreens is largely unknown as no studies on its effect on keratinocytes or melanocytes exist. As such, we aimed to explore the effect of CBD on keratinocyte and melanocyte viability following ultraviolet B (UVB) irradiation. CBD exhibited a dose-dependent protective effect on both keratinocytes and melanocyte viability. Further, since CBD does not demonstrate absorption in the UVB spectra, we speculate that the protective effect is due to reduction in reactive oxygen species. To our knowledge, this is the first study demonstrating the protective effect of CBD on keratinocytes and melanocytes irradiated with UVB.

Androgen sensitivity gateway to COVID-19 disease severity.

In this communication, we present arguments for androgen sensitivity as a likely determinant of COVID-19 disease severity. The androgen sensitivity model explains why males are more likely to develop severe symptoms while children are ostensibly resistant to infection. Further, the model explains the difference in COVID-19 mortality rates among different ethnicities. Androgen sensitivity is determined by genetic variants of the androgen receptor. The androgen receptor regulates transcription of the transmembrane protease, serine 2 (TMPRSS2), which is required for SARS-CoV-2 infectivity. TMPRSS2 primes the Spike protein of the virus, which has two consequences: diminishing viral recognition by neutralizing antibodies and activating SARS-CoV-2 for virus-cell fusion. Genetic variants that have been associated with androgenetic alopecia, prostate cancer, benign prostatic hyperplasia and polycystic ovary syndrome could be associated with host susceptibility. In addition to theoretical epidemiological and molecular mechanisms, there are reports of high rates of androgenetic alopecia of from hospitalized COVID-19 patients due to severe symptoms. Androgen sensitivity is a likely determinant of COVID-19 disease severity. We believe that the evidence presented in this communication warrants the initiation of trials using anti-androgen agents.

Topical Alpha-1 Adrenergic Receptor Agonist Applied to the Nipple/Areola Complex Improves Female Orgasmic Function.

Background: The impact of nipple sensation and its relationship to sexual function have often been neglected in medical literature. However, several recent studies report the importance of the nipple/areola complex (NAC) in sexual arousal and overall function. The nipple is composed of smooth muscle that can be erected via adrenergic nerves. In two complementary studies, we demonstrate that stimulation of the alpha-1 adrenergic receptor in the NAC with topical adrenergic agents can initiate erection of the nipple, increase NAC sensitivity, and improve sexual function. Materials and Methods: Thirteen breast surgery patients with nipple sensitivity loss were recruited to an unblinded study of topical phenylephrine hydrochloride. Sensitivity to pressure was measured before and after the application of the intervention to the NAC. In a second pilot study, 35 women completed a double-blinded placebo-controlled trial of a novel formulation, RJ101, containing a norepinephrine releasing agent. The intervention or placebo was applied to the NAC 30 minutes before sexual activity over the 4-week trial period. The arousal, lubrication, and orgasm domains of the female sexual function index (FSFI) were used to measure sexual function. Results: The application of phenylephrine hydrochloride was shown to increase nipple sensitivity to pressure by an average of 20% in our cohort of 13 breast augmentation patients. In addition, it was shown that intermittent application of the alpha-1 agonist for 8 weeks increased basal NAC sensitivity. In the follow-up pilot study, we demonstrate that stimulation of the NAC with RJ101 produced statistically significant increases versus placebo in the lubrication and orgasm domains of the FSFI, p = 0.0226 and p = 0.0269, respectively. Conclusion: For the first time, we demonstrate that the application of a topical alpha-1 adrenergic receptor agonist or a norepinephrine-releasing agent increases the sensitivity of the NAC and subsequently significantly improves sexual function.

Non-ablative radio frequency for the treatment of androgenetic alopecia.

INTRODUCTION: Medical treatment of androgenetic alopecia (AGA) is mainly limited to pharmacological and surgical interventions. Patients' desire for noninvasive and non-systemic treatments has accelerated research into medical devices that can promote hair growth. Low-level laser therapy (LLLT) was the first such device. However, its success has been limited by contradictory and often controversial efficacy claims. Work previously performed in animal models of AGA has demonstrated the viability of the wound repair mechanism as a potential treatment modality. This study therefore explores the use of a non-ablative radio frequency (RF) device in the treatment of AGA. METHODS: A single blindedstudy compared a non-ablative RF device versus a sham device in 24 men with AGA. Each subject received four treatments over the 12-week study. RESULTS: In this preliminary study of 24 AGA patients treated with a novel RF device, we demonstrated that 54% showed a clinical response. Furthermore, among patients that underwent four or more treatment sessions, 40% experienced a 30% or more increase in hair counts compared to baseline. CONCLUSIONS: If validated in a larger cohort, non-ablative RF may prove to be an important clinical tool in the treatment of AGA.

Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation.

In this study we demonstrate the feasibility of direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors following a brief ex vivo stimulation. Cytokine-secreting cells were identified using cell surface "catch" reagents and single cell data were obtained by sorting of individual cytokine-secreting cells into 96 well plates containing lysis buffer followed by analysis using ultrasensitive immunoassays for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). CD8 cells negative for cytokine production, as determined by the cell surface catch reagents were used as negative controls. Furthermore, studies were undertaken to compare the mean fluorescence intensity (MFI) values of cytokine staining by flow cytometry with the quantification of cytokines using the current method. This study demonstrates that it is feasible to quantify cytokines from individual primary cells. A shift from qualitative to quantitative determinations of cytokine protein levels in single cells will permit more precise and reproducible studies of heterogeneity in the immune system and can be accomplished with readily available instrumentation.

Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. METHODOLOGY: Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. CONCLUSIONS: Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice.

Genetically Modified Live Attenuated Leishmania donovani Parasites Induce Innate Immunity through Classical Activation of Macrophages That Direct the Th1 Response in Mice.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4(+) T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1ß, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani-infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4(+) T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Topical cream delivers NB-UVB from sunlight for the treatment of vitiligo.

Ultraviolet-B (UVB) phototherapy for the treatment of vitiligo is an effective first-line choice. However, the cost of multiple doctor visits and the lengthy treatment regimen has resulted in low compliance, limiting access to this safe and effective mode of treatment. Topical Photocil represents an innovative solution to this problem. The drug selectively filters solar radiation to deliver narrow-band UVB to vitiligo lesions. Here, we discuss how this novel topical cream could provide a convenient alternative to artificial light phototherapy.

Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen(-/-) and Ldp27(-/-)) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag-stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow-derived dendritic cells infected with LdCen(-/-) and Ldp27(-/-) parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan-Leishmania species vaccines.

Novel topical cream delivers safe and effective alternative to traditional psoriasis phototherapy.

In today's environment of shrinking reimbursement and coverage for many health care procedures, phototherapy for psoriasis has experienced a major decline. Once hailed as the cornerstone of psoriasis therapy, the increasing cost and demanding treatment regimen has resulted in low compliance, limiting access to this safe and effective mode of treatment. We have previously reported on the development and in vitro evaluation of a topical cream that selectively filters solar radiation to deliver narrow-band ultraviolet B. Here, we present the results of a pilot study in psoriasis patients. After an average of 38 sessions, all patients in the treatment arm responded to therapy. In particular, 43% of the treatment group experienced complete clearance and the remainder experienced at a minimum 50% lesion clearance. In contrast, none of the patients in the placebo arm experienced more than 20% lesion clearance. Our preliminary results demonstrate that the novel topical cream could provide a safe, effective, and convenient alternative to artificial light phototherapy.

Novel topical cream delivers safe and effective sunlight therapy for vitiligo by selectively filtering damaging ultraviolet radiation.

Numerous studies have demonstrated that natural sunlight therapy at the Dead Sea provides therapeutic efficacy for vitiligo patients on par with artificial broadband and narrowband ultraviolet B (UVB) phototherapy; however, similar treatments at locals at or above sea level fail due to the development of erythema prior to sufficient therapeutic dosage. We conducted a pilot study at sea level to assess the efficacy of a novel topical cream that selectively filters nontherapeutic wavelengths of UVB from natural sunlight and delivers treatment for acrofacial vitiligo. In our pilot study, after an average of 11 weeks of treatment, all patients in the treatment arm responded to therapy. In particular, 28% of the treatment group had 70% surface area repigmentation, 28% had 50% repigmentation, and 44% had 30-40% repigmentation. In contrast, only 10% of the patients in the placebo arm had 20% repigmentation. Our results demonstrate that the novel topical cream can provide a safe and effective alternative to artificial light phototherapy.

In vitro evaluation of a novel topical cream for vitiligo and psoriasis that selectively delivers NB-UVB therapy when exposed to sunlight.

Ultraviolet-B (UVB) phototherapy is a well-established mode of treatment for several types of dermatological disease. For psoriasis and vitiligo, narrow band UVB (NB-UVB) phototherapy is an effective therapy, demonstrating greater efficacy and safety compared to broadband UVB or psoralen plus UVA treatments. While the treatment efficacy of NB-UVB artificial light sources is well documented, the long term time and cost commitment of the therapy remains a barrier to treatment adherence. Natural sunlight is an ideal source of accessible UVB radiation; however, exposure to natural sunlight generally results in erythema prior to the accumulation of sufficient dosage of therapeutic wavelengths of UVB. This communication describes a novel topical cream designed to selectively deliver NB-UVB therapy when exposed to sunlight. The topical cream when combined with natural sunlight could offer patients a more convenient phototherapy option for psoriasis and vitiligo, potentially increasing patient compliance.

Cytopenia and leukocyte recovery shape cytokine fluctuations after myeloablative allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation is associated with profound changes in levels of various cytokines. Emphasis has been placed on conditioning-associated mucosal damage and neutropenia and associated bacterial translocation as the initiating conditions predisposing to acute graft-versus-host disease. The post-transplant period is, however, also associated with increases in certain homeostatic cytokines. It is unclear how much the homeostatic drive to lymphocyte recovery and the production of cytokines from the engrafting donor immune system determine cytokine fluctuations in the peri- and immediate post-transplant period. The aim of this study was to examine the contributions of the conditioning regimen, donor engraftment, infections, and graft-versus-host disease to fluctuations in cytokines involved in homeostasis and inflammation. DESIGN AND METHODS: We examined the levels of 33 cytokines in relation to peri- and post-transplant events such as conditioning regimen, chimerism, and acute graft-versus-host disease in myeloablative, non-T cell-replete HLA-identical sibling donor stem cell transplantation for hematologic malignancies. RESULTS: We identified two cytokine storms. The first occurred following conditioning and reached peak levels when all the leukocytes were at their lowest concentrations. The second cytokine storm occurred concurrently with hematopoietic reconstitution and subsided with the achievement of full donor lymphocyte chimerism. CONCLUSIONS: Our results indicate that both recipient-related and donor-related factors contribute to the changes in cytokine levels in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using plasma samples drawn from patients enrolled in the ClinicalTrials.gov-registered trials NCT00467961 and NCT00378534.

Robust expansion of viral antigen-specific CD4+ and CD8+ T cells for adoptive T cell therapy using gene-modified activated T cells as antigen presenting cells.

Cytomegalovirus (CMV) reactivation after stem cell transplantation can be treated with CMV-specific T cells, but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV-specific T cells, we evaluated gene-modified activated T cells [antigen presenting T cells (T-APCs)] as a reliable and easily produced source of APCs to boost CD4+ and CD8+ T-cell responses against the immunodominant CMV antigen pp65. T-APCs expressing the full-length immunodominant CMV pp65 gene were used to stimulate the expansion of autologous T cells. After 10 to 14 days, the T cell lines were tested for antigen specificity by using the flow cytometric intracellular detection of interferon-gamma after stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. Under optimal conditions, this technique induced a median 766-fold and a 652-fold expansion of pp65-specific CD4+ and CD8+ responder cells, respectively, in 15 T cell lines. In 13 of 15 T cell lines, over 10 antigen-specific CD4+ plus CD8+ T cells were generated starting with only 5x10 peripheral blood mononuclear cells, representing an over 3-log increase. These data indicate that T-APCs efficiently boost pp65-specific CD4+ and CD8+ T cell numbers to clinically useful levels. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3-week culture period using only one stimulation of antigen.

LINGO-1 negatively regulates myelination by oligodendrocytes.

The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.

TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration.

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration.

Association and colocalization of G protein alpha subunits and Purkinje cell protein 2 (Pcp2) in mammalian cerebellum.

Previously, we have demonstrated a novel interaction between Galpha(o) protein and Purkinje cell protein-2 (Pcp2, also known as L7) in vitro and in transfected cells (Luo and Denker [1999] J. Biol. Chem. 274:10685-10688). Pcp2 is uniquely expressed in cerebellar Purkinje cells and in retinal bipolar neurons, and it may function as a cell-type specific modulator for G protein-mediated cell signaling. This interaction has been further evaluated in the present studies. Coimmunoprecipitation experiments reveal that Pcp2 associates with Galpha(o) in vivo in mouse cerebellum and eye extract. Pcp2 also associate with Galpha(i2) in the cerebellum. No detectable associations of Pcp2 with Galpha(z) and Galpha(q) subunits are observed. The association of Galpha(o) and Pcp2 is detected at postnatal day 1 (P1), and the association remains stable from day 3 (P3) until adulthood. Further, immunofluorescent double labeling and confocal microscopy suggest that Pcp2 and Galpha(o) are colocalized in the distal processes of cerebellar Purkinje cells including axonal endings and dendritic spines. Taken together, these findings indicate colocalization and association of Galpha(o) and Pcp2 in cerebellum and suggest a functional role in regions of synaptic activity.