Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP.

Mutagenic translesion DNA polymerase V (UmuD'2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD'2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD'2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is "rapid" at 37°C (~ 1 to 1.5 h), "slow" at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.

Obese visceral adipose dendritic cells downregulate regulatory T cell development through IL-33.

Regulatory T cells (Tregs) residing in visceral adipose tissue (VAT) play a pivotal role in regulating tissue inflammation and metabolic dysfunction associated with obesity. However, the specific phenotypic and functional characteristics of Tregs in obese VAT, as well as the regulatory mechanisms shaping them, remain elusive. This study demonstrates that obesity selectively reduces Tregs in VAT, characterized by restrained proliferation, heightened PD-1 expression, and diminished ST2 expression. Additionally, obese VAT displays distinctive maturation of dendritic cells (DCs), marked by elevated expressions of MHC-II, CD86, and PD-L1, which are inversely correlated with VAT Tregs. In an in vitro co-culture experiment, only obese VAT DCs, not macrophages or DCs from subcutaneous adipose tissue (SAT) and spleen, result in decreased Treg differentiation and proliferation. Furthermore, Tregs differentiated by obese VAT DCs exhibit distinct characteristics resembling those of Tregs in obese VAT, such as reduced ST2 and IL-10 expression. Mechanistically, obesity lowers IL-33 production in VAT DCs, contributing to the diminished Treg differentiation. These findings collectively underscore the critical role of VAT DCs in modulating Treg generation and shaping Treg phenotype and function during obesity, potentially contributing to the regulation of VAT Treg populations.

Disseminated Histoplasmosis Found in Bone Marrow in a Newly Diagnosed AIDS Patient: A Literature Review and Report of a Rare Case.

Histoplasmosis is a rare fungal infection caused by the dimorphic species Histoplasma (H.) capsulatum, found in the Midwest and Central United States. Infection with H. capsulatum is observed in other regions beyond the Ohio and Mississippi River valley, including Mexico and Central and South America. There have been increasing reports of the disease occurring in Latin America in immunocompromised patients with human immunodeficiency virus (HIV). This case report details clinical findings of disseminated histoplasmosis in an immunocompromised patient, newly diagnosed with acquired immunodeficiency syndrome (AIDS) and initially presenting with sepsis of unclear source. The focus of this case report is the significance of detailed history-taking guiding for an appropriate investigation and recognition of the infectious source and giving insight into the management of disseminated histoplasmosis in the outpatient and inpatient settings.

Correction: Disseminated Histoplasmosis Found in Bone Marrow in a Newly Diagnosed AIDS Patient: A Literature Review and Report of a Rare Case.

[This corrects the article DOI: 10.7759/cureus.35417.].

A Mysterious Case of Left Shoulder Pain in AIDS: A Case Report and Literature Review.

Neisseria (N.) gonorrhea is a gram-negative diplococcus and one of the most commonly reported sexually transmitted infections (STIs) in the United States. Disseminated gonococcal infection is a rare but serious complication of N. gonorrhoeae infection that can result in arthritis-dermatitis syndrome or purulent gonococcal arthritis. Co-infection with human immunodeficiency virus (HIV) has been shown to reduce the efficacy of complement recruitment, which may lead to an increased risk of disseminated gonococcal spread. We present a case of a 41-year-old male with concomitant HIV-gonorrhea infection complicated by rare chronic subacute septic arthritis localized to the left shoulder. The patient had a history of HIV, hypertension, and diabetes, and presented with symptoms, including diarrhea, oral thrush, body aches, and fevers. During his hospitalization, the patient developed increasing left shoulder pain, and imaging and joint aspiration revealed N. gonorrhoeae as the causative agent. The patient was treated with appropriate antibiotics and showed improvement. This case highlights the importance of considering disseminated gonococcal infection as a potential complication of N. gonorrhoeae infection, particularly in patients with concomitant HIV infection, and the need for prompt diagnosis and appropriate treatment to prevent complications.

Left-Sided Transudative Chylothorax With Concomitant Chylous Ascites in the Setting of Liver Cirrhosis.

Most chylothoraces are caused by trauma and malignancy, and pleural fluid analysis typically demonstrates an exudative effusion. Transudative chylothorax is a rare manifestation and has only been cited in case reports in the current literature. Here, we present the case of a 59-year-old male with a history of liver cirrhosis secondary to alcohol abuse, chronic kidney disease stage 3a, and hypertension who presented with a left-sided pleural effusion and abdominal ascites. A thoracentesis and abdominal paracentesis were performed, and fluid analyses demonstrated a transudative chylothorax with concomitant chylous ascites. In this review, we aim to highlight a rare case of transudative chylothorax and discuss the pathogenesis and management of this condition.

T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFß) signaling mediate superior tumor regression in an animal model of adoptive cell therapy.

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFß), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFß by transduction with a TGFß dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

Quantitative PET reporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigen.

Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene. Micro-PET imaging using the sr39tk-specific substrate 9-[4-[(18)F]fluoro-3-(hydroxymethyl)-butyl]guanine ([(18)F]FHBG) enabled the detection of transplanted T cells in secondary lymphoid organs of recipient mice over a 3-week period. Tumor responses could be predicted as early as 3 days following AT when a >25-fold increase of micro-PET signal in the spleen and 2-fold increase in lymph nodes (LNs) were observed in mice receiving combined immunotherapy versus control mice. The lower limit of detection was approximately 7 x 10(5) T cells in the spleen and 1 x 10(4) T cells in LNs. Quantification of transplanted T cells in the tumor was hampered by the sr39tk-independent trapping of [(18)F]FHBG within the tumor architecture. These data support the feasibility of using PET to visualize the expansion, homing and persistence of transferred T cells. PET may have significant clinical utility by providing the means to quantify anti-tumor T cells throughout the body and provide early correlates for treatment efficacy.

Immunosensitization with a Bcl-2 small molecule inhibitor.

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.

A Quality-by-Control Approach in Pharmaceutical Continuous Manufacturing of Oral Solid Dosage via Direct Compaction.

The pharmaceutical industry has been undergoing a paradigm shift towards continuous manufacturing, under which novel approaches to real-time product quality assurance have been investigated. A new perspective, entitled Quality-by-Control (QbC), has recently been proposed as an important extension and complementary approach to enable comprehensive Quality-by-Design (QbD) implementation. In this study, a QbC approach was demonstrated for a commercial scale tablet press in a continuous direct compaction process. First, the necessary understanding of the compressibility of a model formulation was obtained under QbD guidance using a pilot scale tablet press, Natoli BLP-16. Second, a data reconciliation strategy was used to reconcile the tablet weight measurement based on this understanding on a commercial scale tablet press, Natoli NP-400. Parameter estimation to monitor and update the material property variance was also considered. Third, a hierarchical three-level control strategy, which addressed the fast process dynamics of the commercial scale tablet press was designed. The strategy consisted of the Level 0 built-in machine control, Level 1 decoupled Proportional Integral Derivative (PID) control loops for tablet weight, pre-compression force, main compression force, and production rate control, and Level 2 data reconciliation of sensor measurements. The effective and reliable performance, which could be demonstrated on the rotary tablet press, confirmed that a QbC approach, based on product and process knowledge and advanced model-based techniques, can ensure robustness and efficiency in pharmaceutical continuous manufacturing.

Enhanced antitumor activity induced by adoptive T-cell transfer and adjunctive use of the histone deacetylase inhibitor LAQ824.

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain.

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain.

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood. We administered non-Ag-loaded, immature dendritic cells (DC) to CD8alpha knockout (KO) mice and studied their anti-CNS tumor immune responses. DC administration induced dramatic antitumor immune protection in CD8alpha KO mice that were challenged with B16 melanoma both s.c. and in the brain. The CNS antitumor immunity was dependent on both CD4+ T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4+ T cell and NK cell expansion in the draining lymph nodes at 6 days postvaccination, which persisted for 2 wk. Finally, DC administration in CD8alpha KO mice was associated with robust infiltration of CD4+ T cells and NK cells into the brain tumor parenchyma. These results represent the first demonstration of a potent innate antitumor immune response against CNS tumors in the absence of toxicity. Thus, non-Ag-loaded, immature DC administration, in the setting of CD8 genetically deficient mice, can induce dramatic antitumor immune responses within the CNS that surpass the effects observed in wild-type mice. Our results suggest that a better understanding of the cross-talk between DC and innate immune cells may provide improved methods to vaccinate patients with tumors located both systemically and within the CNS.

Broad antitumor protection by dendritic cells administered to CD8alpha knock out mice.

Dendritic cell (DC) administration to CD8alpha knock-out (CD8alphaKO) mice results in a strong antigen-non-specific protection to a B16 murine melanoma tumor challenge. This response is mediated by lytic NK cells and cytokine-producing CD4 cells. We aimed to determine the signals that guide tumor targeting of this response. CD8alphaKO mice in the C57BL/6 background received subcutaneous (s.c.) injections of immature DC. Mice were challenged in vivo or assayed for lytic activity in vitro to a panel of syngeneic tumors with different levels of MHC class I expression. These studies support the following conclusions: (1) DC administration to CD8alphaKO mice results in protective in vivo responses to syngeneic tumors from epithelial, neuroectodermal and hematopoietic origin; in vivo protection is independent of the level of MHC classes I and II expression. (2) The in vitro lytic activity of DC-activated NK cells from CD8alphaKO mice has sensitive and insensitive targets, which is independent of the cell lineage or the level of inhibitory self-MHC surface molecules. (3) In sensitive targets a putative activating NK ligand in DC-stimulated NK cells from CD8alphaKO mice signals directly to PI3-K, but is distinct from NKG2D.

Immunosensitization of tumor cells to dendritic cell-activated immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade).

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-kappaB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-kappaB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-alpha, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.