: Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in ...healthy tissues. For many ligand-targeted drug conjugates, a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells. Because disulfide bond reduction is frequently exploited to ensure intracellular drug release, an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design. While the redox properties of folate receptor trafficking endosomes have been previously reported, little is known about the trafficking of prostate-specific membrane antigen (PSMA), a receptor that is experiencing increasing use for drug targeting in humans.
: To obtain this information, we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.
: We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells, and that reduction of its disulfide bond proceeds steadily but incompletely following internalization. The fact that maximal disulfide reduction reaches only ~50%, even after 24 h incubation, suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.
: As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates, it also follows that not all internalizing receptors are translocated through similar intracellular compartments. Taken together, these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.
Objectives/Hypothesis
Folate receptor (FR) expression, although known to be elevated in many types of cancer and inflammatory cells, has not been well characterized in head and neck squamous cell ...carcinoma (HNSCC). We hypothesized that tumor infiltrating inflammatory cells expressing FR‐β could allow fluorescent visualization of HNSCC tumors using folate conjugated dyes even when FR expression in cancer cells is low.
Study Design
Retrospective review of clinical pathologic specimens and in vivo animal study.
Methods
A tissue microarray with tumor and tumor‐free tissue from 22 patients with HNSCC was stained with antibodies to FR‐α and FR‐β. We characterized FR‐β+ cells by examining CD45, CD68, CD206, and transforming growth factor (TGF)‐β expression. To investigate fluorescent imaging, mice with orthotopic tumor xenografts were imaged in vivo after intravenous injections of folate conjugated fluorescein isothiocyanate (folate‐FITC) and were histologically evaluated ex vivo.
Results
All tumor samples demonstrated significant FR‐β staining and negligible FR‐α staining. FR‐β+ cells found in tumors coexpressed CD68 and had increased expression of CD206 and TGF‐β characteristic of tumor‐associated macrophages. In the xenograft models, tumors showed strong in vivo fluorescence after folate‐FITC injection in contrast to surrounding normal tissues. Histologic examination of the xenograft tissue similarly showed folate‐FITC uptake in areas of inflammatory cellular infiltrate.
Conclusions
Although HNSCC tumor cells do not express FR, HNSCC tumors contain a significant population of FR‐β–expressing macrophages. Folate conjugated fluorescent dye is able to specifically target and label tumor xenografts to permit macroscopic fluorescence imaging due to FR‐β expression on the infiltrating inflammatory cells.
Level of Evidence
NA Laryngoscope, 124:E312–E319, 2014
Over three years have passed since the COVID-19 pandemic started. The dangerousness and impact of COVID-19 should definitely not be ignored or underestimated. Other than the symptoms of acute ...infection, the long-term symptoms associated with SARS-CoV-2 infection, which are referred to here as "sequelae of long COVID (LC)", are also a conspicuous global public health concern. Although such sequelae were well-documented, the understanding of and insights regarding LC-related sequelae remain inadequate due to the limitations of previous studies (the follow-up, methodological flaws, heterogeneity among studies, etc.). Notably, robust evidence regarding diagnosis and treatment of certain LC sequelae remain insufficient and has been a stumbling block to better management of these patients. This awkward situation motivated us to conduct this review. Here, we comprehensively reviewed the updated information, particularly focusing on clinical issues. We attempt to provide the latest information regarding LC-related sequelae by systematically reviewing the involvement of main organ systems. We also propose paths for future exploration based on available knowledge and the authors' clinical experience. We believe that these take-home messages will be helpful to gain insights into LC and ultimately benefit clinical practice in treating LC-related sequelae.
Little data is available on the evaluation of the occurrence rates of Epstein-Barr virus (EBV) in saliva and relationship with highly active antiretroviral therapy (HAART) use in HIV/AIDS patients in ...China. We conducted a retrospective cohort study of EBV serological tests for HIV/AIDS patients who were treated in the hospitals for infectious diseases in Wuxi and Shanghai, China from May 2016 to April 2017. The EBV-seropositive samples were identified by ELISA. EBV-specific primers and probes were used for the quantitative detection of viral DNA from saliva via quantitative real-time polymerase chain reaction. CD4 cell counts of the HIV/AIDS patients were detected by a flow cytometry. A total of 372 HIV/AIDS patients were ultimately selected and categorized for this retrospective cohort study. For EBV IgG and IgM, the HIV/AIDS HAART use (H) and non-HAART use (NH) groups had significantly higher seropositive rates than the HIV-negative control group. The HIV/AIDS (NH) group had the highest seropositive rate (IgG, 94.27%; IgM, 68.98%) and the highest incidence of EBV reactivation or infection. For salivary EBV DNA-positive rates and quantities, the HIV/AIDS (H) (73.69%) and the HIV/AIDS (NH) (100%) groups showed significantly higher values than the HIV-negative control group (35.79%, > twofold). Further, the salivary EBV DNA-negative population had significantly higher CD4 cell counts than the EBV DNA-positive population in the HIV/AIDS (H) group and the HIV/AIDS (NH) groups. Thus, HAART use is beneficial in decreasing the EBV salivary shedding in HIV/AIDS patients and indirectly decreases EBV transmission risk.
Antiretroviral therapy (ART) serves as a mainstay in treating human immunodeficiency virus (HIV) infection. An HIV patient is traditionally administered the same ART regimen for life, even if his/her ...viral load has been reduced by several orders of magnitude from the initial viral load. Dose reduction in ART has been clinically explored in a trial‐and‐error manner to reduce side effects and improve ART sustainability. Using artificial intelligence (AI), we have discovered that drugs and doses inputs can be related to viral load reduction through a Parabolic Response Surface (PRS). The AI‐PRS platform can rationally guide a clinically‐actionable approach to identify optimized population‐wide and personalized dosing. In this prospective pilot clinical trial, a combination regimen of tenofovir (TDF), efavirenz (EFV) and lamivudine (3TC) is administered to ten patients. Using AI‐PRS, a 33% reduction in the long‐term TDF maintenance dose (200 mg) is identified compared to standard regimens (300 mg). This regimen keeps the HIV viral load below 40 copies/mL with no relapse during a 144‐week observation period. This study demonstrates that AI‐PRS can potentially serve as a scalable approach to optimize and sustain the long‐term management of HIV as well as a broad spectrum of other indications.
Lifetime antiretroviral therapy (ART) is crucial for HIV/AIDS patients. To find a balance between the ART side effects and the risk of relapse, this prospective pilot clinical study harnesses the parabolic response surface, an artificial intelligence platform, to identify a personalized optimal ART dose. With it, all patients get viral suppression with no relapse during a 144‐week observation period.
Objectives/Hypothesis Folate receptor (FR) expression, although known to be elevated in many types of cancer and inflammatory cells, has not been well characterized in head and neck squamous cell ...carcinoma (HNSCC). We hypothesized that tumor infiltrating inflammatory cells expressing FR-beta could allow fluorescent visualization of HNSCC tumors using folate conjugated dyes even when FR expression in cancer cells is low. Study Design Retrospective review of clinical pathologic specimens and in vivo animal study. Methods A tissue microarray with tumor and tumor-free tissue from 22 patients with HNSCC was stained with antibodies to FR-alpha and FR-beta. We characterized FR-beta+ cells by examining CD45, CD68, CD206, and transforming growth factor (TGF)-beta expression. To investigate fluorescent imaging, mice with orthotopic tumor xenografts were imaged in vivo after intravenous injections of folate conjugated fluorescein isothiocyanate (folate-FITC) and were histologically evaluated ex vivo. Results All tumor samples demonstrated significant FR-beta staining and negligible FR-alpha staining. FR-beta+ cells found in tumors coexpressed CD68 and had increased expression of CD206 and TGF-beta characteristic of tumor-associated macrophages. In the xenograft models, tumors showed strong in vivo fluorescence after folate-FITC injection in contrast to surrounding normal tissues. Histologic examination of the xenograft tissue similarly showed folate-FITC uptake in areas of inflammatory cellular infiltrate. Conclusions Although HNSCC tumor cells do not express FR, HNSCC tumors contain a significant population of FR-beta-expressing macrophages. Folate conjugated fluorescent dye is able to specifically target and label tumor xenografts to permit macroscopic fluorescence imaging due to FR-beta expression on the infiltrating inflammatory cells. Level of Evidence NA Laryngoscope, 124:E312-E319, 2014 PUBLICATION ABSTRACT
We have reported that intracellular glutathione
S-transferases P1 (GSTP1) suppresses LPS (lipopolysaccharide)-induced excessive production of pro-inflammatory factors by inhibiting LPS-stimulated ...MAPKs (mitogen-activated protein kinases) as well as NF-κB activation. But under pathogenic circumstances, physiologic levels of GSTP1 are insufficient to stem pro-inflammatory signaling. Here we show that LPS-induced up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW246.7 cells is significantly reduced by incubating cells with recombinant GSTP1 protein.
In vivo study demonstrates that treatment of mice (i.p.) with recombinant GSTP1 protein effectively suppresses the devastating effects of acute inflammation, which includes reduction of mortality rate of endotoxic shock, alleviation of LPS-induced acute lung injury and abrogation of thioglycolate (TG)-induced peritoneal deposition of leukocytes and polymorphonuclear cells (PMNs). Meanwhile, GSTP1 prevented LPS-induced TNF-α, IL-1β, MCP-1 and NO production
. Further investigation by using confocal microscopy and flow cytometry shows that recombinant GSTP1 protein can be delivered into RAW246.7 cells, mouse peritoneal macrophages and HEK 293 cells suggesting that extracellular GSTP1 protein could be transported across plasma membrane and act as a cytosolic protein. In conclusion our research demonstrates a new finding that increasing cellular GSTP1 level by supplement of recombinant GSTP1 effectively suppresses the devastating effects of acute inflammation.
Heat shock protein 90 (Hsp90) is an abundantly and ubiquitously expressed chaperone with majority of client proteins which act as signal molecules. Transforming growth factor β-activated kinase 1 ...(TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK), and is essential in interleukin-1β (IL-1β) triggered signaling pathways. In the present study, we found that Hsp90 plays an important role in regulating IL-1β signaling by keeping TAK1 stability. The results showed that the specific inhibitor geldanamycin (GA) of Hsp90 dramatically inhibited IL-1β stimulated TAK1-MAPKs and TAK1-nuclear factor-κB (NF-κB) activation, resulting in the decrease of cyclooxygenase-2 (COX-2) protein expression. Silencing Hsp90 expression through RNA interference (RNAi) also down-regulated TAK1, as well as attenuated IL-1β induced phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 MAPKs, and degradation of IκBα. The same results were obtained in T6RZC stable cells which initiated IL-1β-induced cell signaling at the level of the oligomerization and ubquitination of TNF receptor-associated factor 6 (TRAF6). We further found that Hsp90 formed a complex with TAK1 via its N-terminal domain and GA destabilized TAK1 and induced TAK1 degradation through proteasome pathway. Taken together our results demonstrate that Hsp90 regulates IL-1β-induced signaling by interacting with TAK1 and maintaining the stability of TAK1, suggesting that Hsp90 might act as the chaperone of TAK1 in immune and inflammatory responses related with IL-1 signal cascades.
Ever since the concept of alternatively activated macrophages (AAMs or M2-Macrophages) was proposed, their prominent roles in the pathology of tumor progression, allergic inflammatory disorders and ...fibrotic diseases have been well-documented. Consequently, AAM has become a prime target for therapeutic interventions, but the question is how to develop a more effective and more specific AAM-directed therapeutic approach. Our lab unexpectedly discovered that a subpopulation of macrophages isolated from sites of inflammation expresses the β isoform of folate receptor (FR-β), whose expression has also been found to be elevated the in the lung tissue from mice with allergic asthma that is caused or aggravated by a massive accumulation of AAMs. So our central hypothesis is that FR-β could serve as a marker for AAM and enable the selective targeting of AAM in vivo. To test this hypothesis, we adopt this well-established mouse model of allergic asthma and found the restricted expression of FR-β on the infiltrated macrophages in the asthmatic lung tissue. Further examination of these FR-β + macrophages demonstrates that they also express a mannose receptor (CD206) and arginase 1, suggesting that they fall into the class of AAM. Moreover, the overexpressed FR-β on these AAMs can be exploited for selective delivery of folate-conjugated imaging agents to diseased tissue. As an extension of folate targeting strategy, we isolated a fully human anti-human FR-β monoclonal antibody, m909, with relatively high affinity and avidity to human FR-β but neither human FR-α nor mouse FR-β. M909 is not only able to efficiently select FR-β-positive synovial macrophages from rheumatoid arthritis patients, but also able to mediate ADCC towards FR-β-positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. Interestingly, performance of FR-β immunohistochemistry (IHC) in a variety of human tumor tissues as well as inflamed tissues revealed that FR-β is not only expressed on several types of malignant cells but also on the tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which is consistent with the animal data that FR-β acts as a marker for macrophages with alternatively activated phenotype. With this novel monoclonal antibody m909, we further showed that FR-β expression is restricted to the classic CD14highCD16 - monocytes but no other cells in the human peripheral blood. However, few, if any, FR+ circulating blood monocytes were found in mice (neither Balb/c nor C57BL/6J). Our findings suggest that FR-β could be an ideal target molecule for manipulating AAM, and folate ligation can facilitate delivery of attached compounds into AAM to minimize off-target toxicity.
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