Terminally Exhausted CD8+ T Cells Resistant to PD-1 Blockade Promote Generation and Maintenance of Aggressive Cancer Stem Cells.

SIGNIFICANCE: Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.

Introduction: Non-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient's response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers. Methods: Peripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton's lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways. Results: We observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1ß are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1ß reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1ß induced STAT1/STAT3/NF-κß signaling axis as a targeted cascade to promote early drug resistance in cancer cells. Conclusion: Our data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients.

TLR induced IL-27 plays host-protective role against B16BL6 melanoma in C57BL/6 mice.

Elicitation of the tumor-eliminating immune response is a major challenge, as macrophages- constituting a major component of solid tumor mass- play important roles in development, maintenance and tumor regression. The macrophage-expressed Toll-Like Receptors (TLRs) enhance macrophage function and their ability to activate T cells via secretion of cytokines, which may help in tumor regression. IL-27, a member of the IL-12 family of cytokines, is shown to exhibit anti-tumor and anti-angiogenic activities. Herein, we developed B16BL6 melanoma model in C75BL/6 mouse to dissect the crosstalk between TLRs and IL-27 in tumors. We report existence of a novel TLR- IL-27 feed-forward loop, whereby TLRs and IL-27 up-regulated each other's expression, which we found perturbed during melanoma tumorigenesis. Intra-tumoral injection of Imiquimod, a TLR7/8 ligand, reduced the tumor burden; the anti-tumor effect was reversed upon IL-27 and IL-27R silencing by intra-tumorally administered, lentivirally expressed IL-27 and IL-27R shRNA. The reduced tumor growth was accompanied by significantly fewer Treg cells but increased IFN-γ and granzyme B expression by CD8+ T cells. These data indicate the preventative role for TLR-induced IL-27 in aggressive and highly invasive melanoma.

NLGP regulates RGS5-TGFß axis to promote pericyte-dependent vascular normalization during restricted tumor growth.

Altered RGS5-associated intracellular pericyte signaling and its abnormal crosstalk with endothelial cells (ECs) result chaotic tumor-vasculature, prevent effective drug delivery, promote immune-evasion and many more to ensure ultimate tumor progression. Moreover, the frequency of lethal-RGS5high  pericytes within tumor was found to increase with disease progression, which signifies the presence of altered cell death pathway within tumor microenvironment (TME). In this study, we checked whether and how neem leaf glycoprotein (NLGP)-immunotherapy-mediated tumor growth restriction is associated with modification of pericytes' signaling, functions and its interaction with ECs. Analysis of pericytes isolated from tumors of NLGP treated mice suggested that NLGP treatment promotes apoptosis of NG2+ RGS5high -fuctionally altered pericytes by downregulating intra-tumoral TGFß, along with maintenance of more matured RGS5neg  pericytes. NLGP-mediated inhibition of TGFß within TME rescues binding of RGS5 with Gαi and thereby termination of PI3K-AKT mediated survival signaling by downregulating Bcl2 and initiating pJNK mediated apoptosis. Limited availability of TGFß also prevents complex-formation between RGS5 and Smad2 and rapid RGS5 nuclear translocation to mitigate alternate immunoregulatory functions of RGS5high  tumor-pericytes. We also observed binding of Ang1 from pericytes with Tie2 on ECs in NLGP-treated tumor, which support re-association of pericytes with endothelium and subsequent vessel stabilization. Furthermore, NLGP-therapy- associated RGS5 deficiency relieved CD4+  and CD8+ T cells from anergy by regulating 'alternate-APC-like' immunomodulatory characters of tumor-pericytes. Taken together, present study described the mechanisms of NLGP's effectiveness in normalizing tumor-vasculature by chiefly modulating pericyte-biology and EC-pericyte interactions in tumor-host to further strengthen its translational potential as single modality treatment.

Neuropsychiatric Manifestations of Autoimmune Encephalitis in a Tertiary Hospital: A Case Series and Current Perspectives.

Importance: Autoimmune encephalitis (AE) refers to a group of neuropsychiatric conditions associated with specific circulating autoantibodies directed against synaptic receptors, neuronal cell surface proteins, and intracellular targets. Increased recognition of these disorders is of value, as affected patients prominently display cognitive impairment, behavioral disturbances, and seizures requiring multidisciplinary teams, with early recognition often impacting prognosis.Observations: This case series is based on a retrospective record review of adult patients diagnosed with AE between January 1, 2010- January 1, 2020. Cases 1 and 2, demonstrating anti- N-methyl-d-aspartate receptor (NMDAR) encephalitis with initial manifestations of neurologic and psychiatric symptoms, correlate with the literature describing a higher prevalence of this condition in young women. Case 3, despite being seronegative, exhibited classic features of anti-NMDAR encephalitis. Case 4 demonstrates a classic presentation of anti-leucine-rich glioma-inactivated 1 (LGI1) encephalitis: a middle-aged male with psychosis, altered mentation, and epilepsy. Case 5 had a more indolent neuropsychiatric presentation with mild elevation of N-type voltage-gated potassium channel (VGKC) antibody. Case 6, with glutamic acid decarboxylase 65 (GAD65) antibody, was an elderly female with speech dysfunction and altered mentation, and case 7 was an elderly male with GAD65 antibody who had stiff-person syndrome, ataxia, cognitive decline, and thymoma.Conclusions: This retrospective case series describes the clinical details of 7 individuals with AE and overlapping neuropsychiatric symptoms. This series is limited in scope, with a small number of cases and observational findings, which prevents specific conclusions from being drawn. Despite this limitation, the present article explores the nuances of variable presentations of this disease to inform better interdisciplinary management and emphasize the gap areas that need rigorous research.

Pseudodelirium: Psychiatric Conditions to Consider on the Differential for Delirium.

OBJECTIVE: The phenotypes of several psychiatric conditions can very closely resemble delirium; the authors describe such presentations as pseudodelirium. However, because the clinical management of these conditions differs markedly from that of delirium, prompt differentiation is essential. The authors provide an educational review to assist clinicians in identifying and managing psychiatric conditions that may be especially challenging to differentiate from delirium. METHODS: Based on clinical experience, the authors identified four psychiatric conditions as among the most difficult to differentiate from delirium: disorganized psychosis, Ganser syndrome, delirious mania, and catatonia. An overview of each condition, description of clinical features, differentiation of specific phenotypes from delirium, and review of clinical management are also provided. RESULTS: The thought and behavioral disorganization in disorganized psychosis can be mistaken for the clouded sensorium and behavioral dysregulation encountered in delirium. The fluctuating alertness and apparent confusion in Ganser syndrome resemble delirium's altered arousal and cognitive features. As its name suggests, delirious mania presents as a mixture of hyperactive delirium and mania; additional features may include psychosis, autonomic activation, and catatonia. Both delirium and catatonia have hypokinetic and hyperkinetic variants, and the two syndromes can also co-occur. CONCLUSIONS: The clinical presentations of several psychiatric conditions can blend with the phenotype of delirium, at times even co-occurring with it. Detailed evaluation is often required to differentiate such instances of pseudodelirium from delirium proper.

A Wasted Opportunity During a Pandemic: The Foreign Medical Graduates in the USA.

In this brief note from the field, we address an essential issue of non-inclusion of Foreign Medical Graduates (FMG) practicing in the US into the healthcare disaster response in the current pandemic. Because FMGs represent a significant share of the entire country's physician workforce, it seems not prudent to ignore the need to address the current immigration barriers affecting the crucial healthcare needs during this pandemic. Being subjects of the ongoing complex bureaucracy complicated by recent anti-immigrant steps, FMGs that practice for years on temporary (H1B) visas cannot fully join COVID-19 forces. In addition, these physicians face multiple challenges related to their health protection, protection of their immediate family, job security, and the potential risk of being deported. We believe that physicians' immigration status should no longer be disregarded outside of academic interest. It carries the same importance as other public health issues, especially in severe healthcare crises like this pandemic.

Deletion of Glutamine Synthetase Gene Disrupts the Survivability and Infectivity of Leishmania donovani.

Glutamine synthetase (GS) is one of the most important metabolic enzymes which catalyzes ligation of glutamate and ammonia to form glutamine. Previous studies from our lab had revealed significant differences in parasite and host GS enzyme which warranted us to further work on its relevance in parasite. To analyze glutamine synthetase function in Leishmania, we generated GS overexpressors and knockout mutants and evaluated their ability to grow in vitro in monocyte differentiated macrophage and in vivo by infections in BALB/c mice. GS knocked out strain showed significant growth retardation with delayed cell cycle progression and morphological alteration. Null mutants exhibited attenuated infectivity both in in vitro and in vivo experiments and the effect was reverted back when infected with GS complemented parasites. This indicated that the alterations in phenotype observed were indeed due to GS knockout. GS knockout also made the parasite increasingly sensitive to Miltefosine. Detailed investigation of mode of parasite death upon Miltefosine treatment by dual staining with Annexin-V conjugated FITC and propidium iodide, pointed towards apoptotic or necrotic mode of cell death. This is the first report to confirm that GS is essential for the survivability and infectivity of Leishmania donovani, and can be exploited as a potential drug-target.

Cytokines in the generation and function of regulatory T cell subsets in leishmaniasis.

CD4+ T regulatory cells (Tregs) are a group of T lymphocytes that maintain self-tolerance and protect the host from inflammation-induced tissue damage. An interacting network of cytokines and transcription factors influence the origin, differentiation, and function of the Tregs in primary and secondary lymphoid organs. However, following antigenic stimulation, it can also be induced at the sites of infection. Immune cell resident microbial pathogens, such as Leishmania, employ varieties of mechanisms to promote the suppressive functions of Tregs for protective evasion from the host immune system. This establishes a state of immune unresponsiveness in the host, exacerbating the disease in Leishmania infection. Elimination of Leishmania pathogens is accomplished with a strong pro-inflammatory response accompanied by the release of host protective cytokines such as Interleukin-2 (IL-2), Interferon-gamma (IFN-γ), and Tumor necrosis factor-alpha (TNF-α), which functions through suppression of Tregs or making the effector cells recalcitrant to Treg mediated suppression. Nevertheless, during chronic infection, the persistence of unwarranted pro-inflammatory cytokines can trigger self-tissue damage. Tregs limit the consequence of chronic inflammation to restrict self-harm suggesting its mutually opposing role in host protection. Furthermore, Tregs function to prevent complete parasite clearance to provide long-term immunity to re-infection. This review summarizes the roles of pro-inflammatory and anti-inflammatory cytokines involved in the homing, activation, differentiation, and suppression of Tregs in the course of Leishmania infection. We also suggest cytokines that can be modulated as potential therapeutic targets to treat Leishmania infection.

A mushroom derived 'carbohydrate-fraction' reinstates host-immunity and protects from Leishmania donovani infection.

The anti-leishmanial effect of the 'carbohydrate-fraction', isolated from an edible mushroom Astraeus hygrometricus, was evaluated against Leishmania donovani infection both in vitro and in vivo. Ahf-Car induced the expression of inducible nitric oxide synthase 2 (iNOS2) and pro-inflammatory cytokines like TNF-α and IL-12, with subsequent downregulation of the anti-inflammatory cytokines as TGF-ß and IL-10, in vitro and in vivo along with a remarkable increase in the expressions of IL-6, IL-1ß, IFN-γ and IRFs, IRF-7 and IRF-8 in vivo. Ahf-Car also reduced the parasite burden in the spleen and liver dose-dependently with a simultaneous proliferation of Ly6C+ cells in the bone marrow of Leishmania-infected experimental animals. It also increased the monocyte population dose-dependently and the expression of the myeloid transcription factor PU.1, in vivo, which presumably signifies the expansion of protective macrophages. Thus, Ahf-Car might be a potent anti-leishmanial lead with unique and effective adjuvant capacity.

How to identify and manage non-response to clozapine?

Clozapine is the only approved treatment for Treatment-Refractory Schizophrenia. Here we describe a case series of three hospitalized patients with clozapine nonresponse. One of them responded to clozapine after dose adjustments were made for an interaction between clozapine and ciprofloxacin. The other two cases remained clozapine nonresponders despite optimizing clozapine treatment. However, both these patients responded to other antipsychotic medications (APMs) with better tolerability than observed with clozapine treatment. This case series focuses on diagnosing a genuine from a pseudo-nonresponse to clozapine and to consider other APMs in genuine nonresponse before switching to invasive interventions, such as ECT.

Statistical properties of spectral fluctuations of N interacting bosons in a harmonic trap.

Spectral fluctuations of a system of N weakly interacting bosons in an isotropic harmonic trap are studied, with the focus on the deviations from Poisson spectral statistics, typical of a quantum integrable systems. We have utilized the ideas formulated by Makino et al. [Phys. Rev. E 67, 066205 (2003)1063-651X10.1103/PhysRevE.67.066205] who have extended the approach of Berry and Robnik [J. Phys. A 17, 2413 (1984)JPHAC50305-447010.1088/0305-4470/17/12/013]. Earlier investigations of the Berry conjecture [Proc. R. Soc. London, Ser. A 356, 375 (1977)1364-502110.1098/rspa.1977.0140] of Poisson spectral statistics mainly considered quantum systems whose classical counterparts are integrable. However, the system of N weakly interacting bosons in the external trap has no classical counterpart. Also, it is a realistic and experimentally achievable system with close relation to Bose-Einstein condensation. Thus, a stringent analysis of the applicability of the Berry conjecture to this kind of systems is indeed required. We observe that for small boson number, the system is close to integrability and the nearest-neighbor level spacing distribution and the level number variance exhibit deviations from Poisson statistics similar to those of rational rectangular billiards.

Psychosis and seizure disorder: challenges in diagnosis and treatment.

Psychosis temporally related to seizure episodes has been a recognized entity with much clinical attention, yet there are less clear guidelines for treatment. Presence of perceptual disturbances and cognitive impairment symptoms make the differentiation between primary psychoses and seizure- related psychoses blurred. Moreover, there are only a few reported cases describing clinical presentation, diagnostic dilemma and treatment challenges when these two entities are present at the same time, with overlapping symptomatology. We describe such a case where the presence of these two problems complicated the diagnoses and the patient's subsequent management required a very intricate collaboration between psychiatry and neurology. In addition, we review available published articles including case reports, studies, and review articles regarding the diagnosis and treatment of this complicated clinical presentation. Some of the analyses were reviewed in detail and resulting outcomes are discussed. Finally, we review the diagnostic and treatment guidelines in the context of the presenting case.

Spectral fluctuation and correlation structure of δ(n) statistics in the spectra of interacting trapped bosons.

It is a well-known fact that the statistical behaviors of level fluctuation and level correlation in the energy-level spectra are the most efficient tool to characterize quantum chaos in nonintegrable quantum systems. The system of interacting trapped bosons is a complex system where the low-lying energy levels are highly influenced by the level repulsion. In this case, interatomic interaction is a dominating fact with strong level correlation between distant levels. Here we numerically calculate the correlation function, number variance, and Dyson-Mehta least-square deviation for the low-lying levels for a few thousand interacting trapped bosons, and our data show good analogy with the Gaussian orthogonal ensemble (GOE) results with a signature of chaos. In the next part of our study, the energy spectrum of these low-lying levels is considered as a discrete signal and the fluctuation of the excitation energy is considered as discrete time series. Then we calculate numerically the height-height correlation function for different order of momentum. In our study logarithmic correlation structure is found instead of multiscaling structure, and we observe that spectral statistics are compatible with those of GOE.

Spectral fluctuation and 1/fα noise in the energy level statistics of interacting trapped bosons.

It has been recently shown numerically that the transition from integrability to chaos in quantum systems and the corresponding spectral fluctuations are characterized by 1/f^{α} noise with 1≤α≤2. The system of interacting trapped bosons is inhomogeneous and complex. The presence of an external harmonic trap makes it more interesting as, in the atomic trap, the bosons occupy partly degenerate single-particle states. Earlier theoretical and experimental results show that at zero temperature the low-lying levels are of a collective nature and high-lying excitations are of a single-particle nature. We observe that for few bosons, the P(s) distribution shows the Shnirelman peak, which exhibits a large number of quasidegenerate states. For a large number of bosons the low-lying levels are strongly affected by the interatomic interaction, and the corresponding level fluctuation shows a transition to a Wigner distribution with an increase in particle number. It does not follow Gaussian orthogonal ensemble random matrix predictions. For high-lying levels we observe the uncorrelated Poisson distribution. Thus it may be a very realistic system to prove that 1/f^{α} noise is ubiquitous in nature.

Reduction of 68Ge activity containing liquid waste from 68Ga PET chemistry in nuclear medicine and radiopharmacy by solidification.

PET with 68Ga from the TiO2- or SnO2- based 68Ge/68Ga generators is of increasing interest for PET imaging in nuclear medicine. In general, radionuclidic purity (68Ge vs. 68Ga activity) of the eluate of these generators varies between 0.01 and 0.001%. Liquid waste containing low amounts of 68Ge activity is produced by eluting the 68Ge/68Ga generators and residues from PET chemistry. Since clearance level of 68Ge activity in waste may not exceed 10 Bq/g, as stated by European Directive 96/29/EURATOM, our purpose was to reduce 68Ge activity in solution from >10 kBq/g to <10 Bq/g; which implies the solution can be discarded as regular waste. Most efficient method to reduce the 68Ge activity is by sorption of TiO2 or Fe2O3 and subsequent centrifugation. The required 10 Bq per mL level of 68Ge activity in waste was reached by Fe2O3 logarithmically, whereas with TiO2 asymptotically. The procedure with Fe2O3 eliminates ≥90% of the 68Ge activity per treatment. Eventually, to simplify the processing a recirculation system was used to investigate 68Ge activity sorption on TiO2, Fe2O3 or Zeolite. Zeolite was introduced for its high sorption at low pH, therefore 68Ge activity containing waste could directly be used without further interventions. 68Ge activity containing liquid waste at different HCl concentrations (0.05-1.0 M HCl), was recirculated at 1 mL/min. With Zeolite in the recirculation system, 68Ge activity showed highest sorption.

Extraction of Hg(I), Hg(II) and methylmercury using polyethylene glycol based aqueous biphasic system.

Extraction behavior of three different salts of mercury, HgCl(2), Hg(2)Cl(2) and CH(3)HgCl, were studied in a polyethylene glycol based aqueous biphasic extraction system (ABS). The results were compared with extraction pattern in a liquid-liquid system using methylisobutyl ketone (MIBK), against HCl and a solid chelating ion exchanger, Chelex 100. The results showed a variable extent of partitioning of different salts of mercury using MIBK and Chelex 100 as ion exchangers. However, when ABS was employed, complete extractions of all the three species of mercury were achieved irrespective of their chemical form.

In situ gamma-radiation: one-step environmentally benign method to produce gold-palladium bimetallic nanoparticles.

Synthesis of gold-palladium bimetallic nanoparticles using in situ radioactivity from (198)Au isotope is reported in this paper. Gold solution spiked with (198)Au(III) has been mixed with PdCl2 solution in measured proportions in 50% polyethylene glycol solution. Au(III) and Pd(II) are reduced, and nanoparticles are formed due to radiolysis of the polymer solution. The solution has been characterized by UV-visible spectra, and the actual size has been determined using transmission electron microscopy in conjugation with energy dispersive X-ray measurements.

Complexation study on no-carrier-added astatine with insulin: a candidate radiopharmaceutical.

No-carrier-added astatine radionuclides produced in the (7)Li-irradiated lead matrix were separated from bulk lead nitrate target by complexing At with insulin, followed by dialysis. The method offers simultaneous separation of At from lead as well as its complexation with insulin. The At-insulin complex might be a potential radiopharmaceutical in the treatment of hepatocellular carcinoma. The stability of At-insulin complex was checked by dialysis against deionized water and Ringer lactate (RL) solution. It has been found that the half-life of At-insulin complex is about approximately 12h, when dialyzed against deionized water and is only 6h, when dialyzed against RL solution having the same composition as blood serum. The 6h half-life of this Insulin-At complex is perfect for killing cancer cells from external cell surfaces as the half-life of internalization of insulin molecule inside the cell is 7-12h.