Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRß. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

Carbapenemase producing Gram negative bacteria: Review of resistance and detection methods.

Gram negative bacilli that are carbapenem resistant have emerged and are spreading worldwide. Infections caused by carbapenem resistant isolates posses a significant threat due to their high morbidity and mortality rates. Carbapenemases production by multi-drug resistant pathogens severely restricts treatment choices for illnesses caused by bacteria that are resistant to both carbapenems and majority of ß-lactam antibiotics. Various phenotypic and genotypic methods for identification can distinguish between different classes of carbapenemase and identify pathogens that are resistant to carbapenems. The establishment of a quick, accurate and reliable test for identifying the clinical strains that produce the carbapenemase enzyme is essential for optimum diagnosis of microbial pathogens and management of the global rise in the prevalence of carbapenemase producing bacterial strains. The aim of this review was to summarize the mechanisms of carbapenem resistance and to provide an overview of different carbapenemase detection methods for carbapenem resistant Gram negative bacilli.

A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo.

Protein-based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non-toxic analogue of lipopolysaccharide. In mice, in comparison with non-formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T-cell responses to HIV gag-p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC-205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen-specific T-cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL-12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T-cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.

Unusual Presentation and Difficult to Diagnose: A Case of Malaria With Negative Thick and Thin Giemsa Stain Smear Tests.

Malaria is a parasitic disease that is spread by the bite of an Anopheles mosquito carrying the infection. Microscopic analysis of thick and thin Giemsa-stained smears is the gold standard for diagnosis. If the initial test is negative, but clinical suspicion is high, further smears are required. A 25-year-old man presented with abdominal distension, cough, and a seven-day fever. In addition, the patient developed pleural effusions and ascites. The thick and thin smear tests for malaria and all other fever testing came out negative. Plasmodium vivax was later identified by reverse transcription polymerase chain reaction (RT-PCR). There was a considerable improvement once the anti-malarial medicine was started. It was difficult to diagnose him because pleural effusion and ascites are unusual for someone with malaria. Furthermore, several Giemsa stain smears and malaria quick diagnostic tests were negative, and only a few labs in our country performed RT-PCR.

A Rare Case of Arterial and Venous Thrombosis in COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has brought attention to the significant risk of thrombotic complications in infected individuals. We present a rare case of a 64-year-old male with COVID-19 who developed bilateral deep vein thrombosis (DVT), pulmonary embolism (PE), and thrombus in the thoracic and abdominal aorta. The patient exhibited common symptoms of COVID-19 and required intensive care unit admission due to respiratory failure. Subsequent investigations revealed thrombi in the lower extremities, pulmonary arteries, and aorta. Prompt anticoagulation therapy was initiated, and vascular surgery consultation was sought. This case highlights the increased risk of venous and arterial thrombotic events in COVID-19 patients and emphasizes the importance of comprehensive management strategies. The interplay of various factors in COVID-19 contributes to a prothrombotic state, necessitating a multi-modal approach to address thrombotic complications. Further research is needed to optimize treatment protocols and improve outcomes for COVID-19 patients with thrombotic complications.

Stauffer Syndrome as the Initial Presentation of Advanced Metastatic Prostate Cancer.

Stauffer's syndrome is a paraneoplastic syndrome that has historically been associated with renal cell carcinoma. It is defined by the anicteric elevation of liver enzymes in the absence of liver metastasis, and the reversibility of clinical and biochemical changes upon treatment of the primary pathology. Here, we discuss the rare presentation of Stauffer's syndrome in a patient with advanced metastatic prostate cancer. A 72-year-old male presented with generalized weakness, dizziness, weight loss, and icterus who was incidentally found to have a prostatic enlargement on physical examination. The laboratory investigations and radiographic imaging confirmed the diagnosis of metastatic prostatic cancer without any evidence of mechanical biliary obstruction as confirmed by biopsy and imaging. The cancer had metastasized to pelvic sidewalls, pelvic bones, ribs, urinary bladder, and local lymph nodes. Our case signifies that a high index of suspicion for underlying cancer should be maintained in patients presenting with cholestatic liver dysfunction, with or without jaundice, especially in the absence of a recognizable mechanical etiology of cholestasis.

Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti-human DEC205 monoclonal antibody.

Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.

Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy (CADASIL): A Rare Cause of Transient Ischemic Attack.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is increasingly recognized as an inherited and autosomal dominant arteriopathy of the cerebral vasculature, which is commonly misdiagnosed due to its different modes of presentation. It is characterized by variable manifestations of ischemic episodes, migraine with aura, cognitive deficits, and psychiatric disturbances. CADASIL is caused by a genetic mutation in the NOTCH3 gene, which is present on chromosome 19. The diagnosis of CADASIL can be made by personal and family history, skin biopsy, and magnetic resonance imaging (MRI) of the head showing high-intensity signal lesions, microbleeds, and white matter changes. There are currently no disease-modifying therapies available for CADASIL, and management focuses on reducing risk factors such as diabetes and hypertension and control of symptoms. We present a rare cause of transient ischemic attack (TIA) in a young female who was later diagnosed with CADASIL and aim to highlight rare and inherited causes of TIA and strokes in younger patients.

Ophthalmoparesis and Bilateral Ptosis as a Rare Manifestation of Todd's Phenomenon: Case Report and Review.

Todd's paresis or phenomenon (TP) is a focal weakness in a part of the body after a seizure. Seizure is an abrupt change in behavior caused by the cerebral cortex's electrical hyper-synchronization of neuronal networks. After the seizure, there is usually a transition period from the ictal state to the pre-seizure baseline level of awareness and function, referred to as the postictal period. Postictal symptoms include many systems, including sensory, motor, and psychosis. This phenomenon is named after Robert Bentley Todd, who first described it. Todd's paresis can be confused with other conditions, most commonly a stroke. Postictal ocular manifestation may be accompanied by aphasia or hemiplegia, but isolated gaze palsy is rarely reported. We are reporting a rare and first known isolated ophthalmoparesis and ptosis as postictal findings with no structural abnormalities present in imaging studies and complete resolution over three weeks on its own as an atypical postictal phenomenon. Patients with an underlying structural abnormality of the brain are more susceptible to Todd's phenomenon. Unusual manifestations of Todd's phenomenon are rare but clinically relevant and are decisive in therapeutic decision-making. Our patient presents a rare manifestation of Todd's phenomenon as ptosis and ophthalmoparesis in an elderly male with no underlying structural brain abnormalities that resolved within three weeks. Further research into the causes is needed to distinguish it from a stroke.

Pneumomediastinum and Subcutaneous Emphysema in an Adult Male From Nepal Infected With COVID-19.

Pneumomediastinum and subcutaneous emphysema have been reported in COVID-19 around the world except for Nepal. We report a case of a 44-year-old male infected with COVID-19 who developed pneumomediastinum and subcutaneous emphysema during his eighth day of intubation at the hospital. He was managed with remdesivir, antibiotics, mechanical ventilation, steroid, and heparin following which he recovered well. Barotrauma-related complications are common in COVID-19 and our case highlights the importance of conservative management for such complications and the rarity of such conditions in Nepal.

Drug-Induced Deafness: A Rare Case of Bilateral Sensorineural Hearing Loss Following Speedballing.

Many users of recreational drugs use cocaine and opioids together, often called "speedballing." Hearing loss is a rarely reported adverse effect following recreational drug abuse. Only one case has been reported in history with hearing loss caused by speedballing. Here, we present the case of a 38-year-old female who presented with speedball abuse and new-onset bilateral hearing loss to the emergency department. A computed tomography scan of the head was unremarkable. She was treated with thiamine, folate, multivitamins, and intravenous fluids. The hearing loss improved without any acute intervention. The significance of sudden hearing loss due to recreational drug use is highlighted by this case. Apart from a few animal studies, there is no detailed research explaining the pathophysiology of speedball-induced hearing loss. Further studies and trials are needed to better understand the effects of combined and separate cocaine and opioid use on audiologic physiology.

BNT162b2 mRNA Vaccine Interference with Co-Administration of Tdap Vaccine.

BACKGROUND It is unknown if the efficacy of the coronavirus disease-19 (COVID-19) vaccine is affected by the co-administration of other vaccines. The Centers for Disease Control and Prevention (CDC) has shifted their recommendations recently, allowing for the co-administration of the currently available COVID-19 vaccines with other vaccines. This is based on the experience with non-COVID-19 vaccines, where the immunogenicity and adverse event profiles were generally similar when vaccines are administered simultaneously or alone. CASE REPORT We present a case of a 29-year-old Asian woman who received the first dose of BNT162b2 mRNA vaccine and the tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine at around the same time. BNT162b2 mRNA vaccine and Tdap vaccine were administered into the deltoid region of the left arm and right arm, respectively. We then monitored for immunogenicity. We observed a delay in the development of SARS-CoV-2 Spike (S1) protein antibodies at around 8 weeks after the second dose. CONCLUSIONS Unless warranted, it is important to adhere to current CDC recommendations with regards to the co-administration of vaccines. Although the administration of Tdap with COVID-19 vaccine in our case caused delay in immunogenicity, it did not negate the ability of the BNT162B2 mRNA vaccine to elicit an adequate immune response. The reason for delay in immune response with co-administration of COVID-19 vaccines with other vaccines is unknown and further studies are needed.

Inhibiting LXRα phosphorylation in hematopoietic cells reduces inflammation and attenuates atherosclerosis and obesity in mice.

Atherosclerosis and obesity share pathological features including inflammation mediated by innate and adaptive immune cells. LXRα plays a central role in the transcription of inflammatory and metabolic genes. LXRα is modulated by phosphorylation at serine 196 (LXRα pS196), however, the consequences of LXRα pS196 in hematopoietic cell precursors in atherosclerosis and obesity have not been investigated. To assess the importance of LXRα phosphorylation, bone marrow from LXRα WT and S196A mice was transplanted into Ldlr-/- mice, which were fed a western diet prior to evaluation of atherosclerosis and obesity. Plaques from S196A mice showed reduced inflammatory monocyte recruitment, lipid accumulation, and macrophage proliferation. Expression profiling of CD68+ and T cells from S196A mouse plaques revealed downregulation of pro-inflammatory genes and in the case of CD68+ upregulation of mitochondrial genes characteristic of anti-inflammatory macrophages. Furthermore, S196A mice had lower body weight and less visceral adipose tissue; this was associated with transcriptional reprograming of the adipose tissue macrophages and T cells, and resolution of inflammation resulting in less fat accumulation within adipocytes. Thus, reducing LXRα pS196 in hematopoietic cells attenuates atherosclerosis and obesity by reprogramming the transcriptional activity of LXRα in macrophages and T cells to promote an anti-inflammatory phenotype.

LXRα Phosphorylation in Cardiometabolic Disease: Insight From Mouse Models.

Posttranslational modifications, such as phosphorylation, are a powerful means by which the activity and function of nuclear receptors such as LXRα can be altered. However, despite the established importance of nuclear receptors in maintaining metabolic homeostasis, our understanding of how phosphorylation affects metabolic diseases is limited. The physiological consequences of LXRα phosphorylation have, until recently, been studied only in vitro or nonspecifically in animal models by pharmacologically or genetically altering the enzymes enhancing or inhibiting these modifications. Here we review recent reports on the physiological consequences of modifying LXRα phosphorylation at serine 196 (S196) in cardiometabolic disease, including nonalcoholic fatty liver disease, atherosclerosis, and obesity. A unifying theme from these studies is that LXRα S196 phosphorylation rewires the LXR-modulated transcriptome, which in turn alters physiological response to environmental signals, and that this is largely distinct from the LXR-ligand-dependent action.

Impaired LXRα Phosphorylation Attenuates Progression of Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2.

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

Modulation of Macrophage Gene Expression via Liver X Receptor α Serine 198 Phosphorylation.

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by liver X receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRα serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRα at S198 is nonphosphorylated. In bone marrow-derived macrophages (BMDMs), we also observed induction of CCR7 by ligands that promote nonphosphorylated LXRα S198, and this was lost in LXR-deficient BMDMs. LXRα occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing nonphosphorylated LXRα (RAW-LXRα S198A) compared to RAW264.7 cells expressing wild-type (WT) phosphorylated LXRα (RAW-LXRα WT). Expression profiling of ligand-treated RAW-LXRα S198A cells compared to RAW-LXRα WT cells revealed induction of cell migratory and anti-inflammatory genes and repression of proinflammatory genes. Modeling of LXRα S198 in the nonphosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with the presence of sites for protein interaction. Therefore, gene transcription is regulated by LXRα S198 phosphorylation, including that of antiatherogenic genes such as CCR7.

New monoclonal anti-mouse DC-SIGN antibodies reactive with acetone-fixed cells.

Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone-fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.