Intratumoral injection of schwannoma with attenuated Salmonella typhimurium induces antitumor immunity and controls tumor growth.

Schwannomas are slow-growing benign neoplasms that develop throughout the body causing pain, sensory/motor dysfunction, and death. Because bacterial immunotherapy has been used in the treatment of some malignant neoplasms, we evaluated attenuated Salmonella typhimurium strains as immunotherapies for benign murine schwannomas. Several bacterial strains were tested, including VNP20009, a highly attenuated strain that was previously shown to be safe in human subjects with advanced malignant neoplasms, and a VNP20009 mutant that was altered in motility and other properties that included adherence and invasion of cultured mammalian cells. VNP20009 controlled tumor growth in two murine schwannoma models and induced changes in cytokine and immune effector cell profiles that were consistent with induction of enhanced innate and adaptive host immune responses compared with controls. Intratumoral (i.t.) injection of S. typhimurium led to tumor cell apoptosis, decreased tumor angiogenesis, and lower growth of the injected schwannoma tumors. Invasive VNP20009 was significantly more efficacious than was a noninvasive derivative in controlling the growth of injected tumors. Bacterial treatment apparently induced systemic antitumor immunity in that the growth of rechallenge schwannomas implanted following primary bacterial treatment was also reduced. Checkpoint programmed death-1 (PD-1) blockade induced by systemic administration of anti-PD-1 antibodies controlled tumor growth to the same degree as i.t. injection of S. typhimurium, and together, these two therapies had an additive effect on suppressing schwannoma growth. These experiments represent validation of a bacterial therapy for a benign neoplasm and support development of S. typhimurium VNP20009, potentially in combination with PD-1 inhibition, as a schwannoma immunotherapy.

Cognitive impairment following breast cancer treatments: an umbrella review.

OBJECTIVES: Cancer-related cognitive impairment (CRCI) refers to a cognitive decline associated with cancer or its treatments. While research into CRCI is expanding, evidence remains scattered due to differences in study designs, methodologies, and definitions. The present umbrella review aims to provide a comprehensive overview of the current evidence regarding the impact of different breast cancer therapies on cognitive functioning, with a particular focus on the interplay among objective cognitive deficits (ie, measured with standardized tests), subjective cognitive concerns, (ie, self-reported), and other mediating psycho-physical factors. METHODS: The search was made in Pubmed, Embase, and Scopus for articles published until July 2023, following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis protocol. RESULTS: Chemotherapy and endocrine therapy appear consistently associated with CRCI in patients with breast cancer, primarily affecting memory, attention/concentration, executive functioning, and processing speed. Subjective cognitive concerns were often found weakly or not associated with neuropsychological test results, while overall CRCI seemed consistently associated with psychological distress, fatigue, sleep quality, and inflammatory and biological factors. CONCLUSION: Current evidence suggests that CRCI is common after chemotherapy and endocrine therapy for breast cancer. However, heterogeneity in study designs and the scarcity of studies on more recent treatments such as targeted therapies and immunotherapies, highlight the need for more systematic and harmonized studies, possibly taking into account the complex and multifactorial etiology of CRCI. This may provide valuable insights into CRCI's underlying mechanisms and potential new ways to treat it.

The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system.

The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. Here we identified and analyzed the direct targets of CsrA in the human pathogen Legionella pneumophila. Genome wide transcriptome, proteome and RNA co-immunoprecipitation followed by deep sequencing of a wild type and a csrA mutant strain identified 479 RNAs with potential CsrA interaction sites located in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs. Further analyses revealed that CsrA exhibits a dual regulatory role in virulence as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it also directly regulates the timely expression of over 40 Dot/Icm substrates. CsrA controls its own expression and the stringent response through a regulatory feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum sensing and motility. CsrA is a central player in the carbon, amino acid, fatty acid metabolism and energy transfer and directly affects the biosynthesis of cofactors, vitamins and secondary metabolites. We describe the first L. pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA, the active stabilization of RNA anti-terminator conformations inside a coding sequence preventing Rho-dependent termination of the gap operon through transcriptional polarity effects. This allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Thus the L. pneumophila genome has evolved to acclimate at least five different modes of regulation by CsrA giving it a truly unique position in its life cycle.

Use of Telemedicine to Improve Cognitive Functions and Psychological Well-Being in Patients with Breast Cancer: A Systematic Review of the Current Literature.

The diagnosis and side effects of breast cancer (BC) treatments greatly affect the everyday lives of women suffering from this disease, with relevant psychological and cognitive consequences. Several studies have reported the psychological effects of receiving a diagnosis of BC. Moreover, women undergoing anticancer therapies may exhibit cognitive impairment as a side effect of the treatments. The access to cognitive rehabilitation and psychological treatment for these patients is often limited by resources; women of childbearing age often encounter difficulties in completing rehabilitation programs requiring access to care institutions. Telemedicine, which provides health services using information and communication technologies, is a useful tool to overcome these limitations. In particular, telemedicine may represent an optimal way to guarantee cognitive rehabilitation, psychological support, and recovery to BC patients. Previous studies have reviewed the use of telemedicine to improve psychological well-being in BC patients, and a few have investigated the effect of telerehabilitation on cognitive deficits. This study systematically reviewed the evidence on the cognitive and psychological effects of telemedicine in BC patients. Current evidence suggests that telemedicine may represent a promising tool for the management of some psychological problems experienced by breast cancer patients, but more controlled studies are needed to clarify its effectiveness, especially for cognitive deficits. The results are also discussed in light of the intervening and modulating factors that may mediate both side effect occurrence and the success of the interventions.

Culture of attenuated Salmonella Typhimurium VNP20009 in animal-product-free media does not alter schwannoma growth control.

Schwannomas are slow-growing benign peripheral nerve sheath tumors derived from Schwann-lineage cells that develop in association with NF2-related schwannomatosis (NF2) and schwannomatosis (NF3), as well as spontaneously. Individuals affected with NF2 and NF3 have multiple schwannomas with tumors arising throughout life. Surgical resection, the standard management, is limited in scope and efficacy and is itself associated with significant morbidity. We have previously shown that direct intratumoral injection of attenuated Salmonella Typhimurium (S. Typhimurium), strain VNP20009, showed a potent anti-tumor effect in preclinical NF-2 schwannoma models. The United States Federal Drug Administration (FDA) requires that bacterial products utilized in clinical trials be produced without exposure to animal-derived-products. In this context, we developed, characterized, and tested the antitumor efficacy of an attenuated S. Typhimurium serially passaged in animal-product-free media, naming it VNP20009-AF for "VNP20009-animal-product-free." Our in vitro data did not indicate any significant changes in the viability, motility, or morphology of VNP20009-AF, compared to its parental strain. In vivo efficacy data demonstrated that VNP20009-AF and VNP20009 controlled tumor growth to the same degree in both human NF2-schwannoma xenograft and murine-NF2 schwannoma allograft models. Together, these data support the use of VNP20009-AF for the translation of bacterial schwannoma therapy into clinical trials.

A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.

The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism.

Legionella pneumophila is a gram-negative bacterium that inhabits freshwater ecosystems, where it is present in biofilm or as planktonic form. L. pneumophila is mainly found associated with protozoa, which serve as protection from hostile environments and as replication niche. If inhaled within aerosols, L. pneumophila is also able to infect and replicate in human alveolar macrophages, eventually causing the Legionnaires' disease. The transition between intracellular and extracellular environments triggers a differentiation program in which metabolic as well as morphogenetic changes occur. We here describe the current knowledge on how the different developmental states of this bacterium are regulated, with a particular emphasis on the stringent response activated during the transition from the replicative phase to the infectious phase and the metabolic features going in hand. We propose that the cellular differentiation of this intracellular pathogen is closely associated to key metabolic changes in the bacterium and the host cell, which together have a crucial role in the regulation of L. pneumophila virulence.

A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. IMPORTANCE: The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.

Small RNAs, 5' UTR elements and RNA-binding proteins in intracellular bacteria: impact on metabolism and virulence.

Sequencing-based studies have illuminated increased transcriptional complexity within the genome structure of bacteria and have resulted in the identification of many small regulatory RNAs (sRNA) and a large amount of antisense transcription. It remains an open question whether these sRNAs all indeed play regulatory roles, but their identification led to an exponential increase in studies searching for their function. This allowed to show that sRNAs may modulate virulence gene expression, cellular differentiation, metabolic functions, adaptation to environmental conditions and pathogenesis. In this review we will provide mechanistic insights into how sRNAs bind mRNAs and/or proteins. Furthermore, the important roles of the RNA chaperone Hfq, the CsrA system and the CRISPR RNA will be discussed. We will then focus on sRNAs and 5(') untranslated region (UTR) elements of intracellular bacteria like Chlamydia, Listeria, Legionella, or Salmonella, and place emphasis on those that are expressed during replication in host cells and are implicated in virulence and metabolism. In addition, sRNAs that regulate motility, iron homeostasis, and differentiation or stress responses will be highlighted. Taken together sRNAs constitute key elements in many major regulatory networks governing the intracellular life and virulence of pathogenic bacteria.

Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316.

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.