Vitiligo-like Lesions as a Predictor of Response to Immunotherapy in Non-Small Cell Lung Cancer: Comprehensive Review and Case Series from a University Center.

The reference to vitiligo-like lesions (VLLs) induced by immune checkpoint inhibitors (ICIs) as a valuable predictive marker of treatment success of immunotherapy with ICIs in melanoma has been mentioned in the literature. Its role in non-small cell lung cancer (NSCLC)-treated patients remains a poorly recognized phenomenon with uncertain significance regarding its predictive value. A retrospective, observational, single-center report was performed, with descriptive analysis of clinicopathological and treatment characteristics of patients with stage IV NSCLC who developed ICI-induced VLL between January 2018 and December 2022, contextualized in a comprehensive review of the literature and reported cases regarding this phenomenon. During the first 5 years' experience of ICI use in stage IV NSCLC treatment, three cases of ICI-induced VLLs were diagnosed. In line with the previous reports, two of the three presented cases exhibited treatment response and favorable prognosis. The recognition and understanding of the pathophysiological processes underlying ICI-induced VLLs may represent a promising opportunity to identify a predictive marker of tumor response to ICIs, with impact in treatment selection and patient management. It also may contribute to the recognition of new patterns of molecular expression that could lead to improvements in therapeutic development.

Impact of Immune-Related Adverse Events on Immune Checkpoint Inhibitors Treated Cancer Patients' Survival: Single Center Experience and Literature Review.

Immune-related adverse events have emerged as a new challenge and its correlation with survival remains unclear. The goal of our study was to investigate the effect of irAE on survival outcomes in solid tumor patients receiving ICI treatment. This was a retrospective, single-center study at a university hospital involving patients with malignancy who received immune checkpoint inhibitors. Chart review was performed on each patient, noting any irAE, including new events or worsening of previous autoimmune condition after starting treatment with ICI. A total of 155 patients were included, 118 (76.1%) were male, with median age of 64 years. Median follow up time was 36 months. Seventy patients (45.2%) had at least one irAE. Of all irAE, nine (8.1%) were classified as grade 3 or higher according to the CTCAE version 5.0. There was one death secondary to pneumonitis. Median ICI cycles until first irAE onset was 4 (range: 2-99). The objective response rate was higher for patients who developed irAE (18.7% vs. 9.0%; p = 0.001), as was median overall survival (18 months (95% CI, 8.67-27.32) vs. 10 (95% CI, 3.48-16.52) months; p < 0.016) and progression free survival (10 months (95% CI, 5.44-14.56) vs. 3 months (95% CI, 1.94-4.05); p = 0.000). The risk of death in patients with irAE was 33% lower when compared to patients without such events (hazard ratio (HR): 0.67; 95% CI, 0.46-0.99; p = 0.043). Development of irAE predicted better outcomes, including OS in patients with advanced solid tumors treated with ICI. Further prospective studies are needed to explore and validate this prognostic value.

Chemotherapy: A partnership with immunotherapy in non-small cell lung cancer.

Chemotherapy (CT) and immunotherapy (IO) act synergically in the treatment of non-small cell lung cancer (NSCLC). However, the molecular basis of such interaction is poorly understood. The aim of this review was to explore the mechanisms of CT to potentiate the immune system and, consequently, the action of IO. The most up-to-date knowledge concerning the interaction of CT and IO in NSCLC was reviewed and a bibliographic search was made in PubMed/Medline database, using the mentioned keywords, with preference given to recently published articles in English. In addition to the direct cytotoxic effect, CT affects the immune system leading indirectly to cell death. The immune response triggered by PD-1 inhibition is enhanced by the cytotoxic immunogenic effects of CT. This potentiation phenomenon occurs due to an increase in effector cells relatively to regulatory cells, inhibition of myeloid derived suppressor cells, increased potential for cross-presentation by dendritic cells after the death of tumor cells or blocking the STAT6 pathway to increase dendritic cell activity. In conclusion, the effects of CT on the immune system work in synergy with the actions of IO, transforming "cold" tumors into "hot" tumors, which are more visible to the immune system.

Immunotherapy in Elderly Patients-Single-Center Experience.

Cancer management faces a substantial challenge posed by the aging demographic. Aging is marked by accumulated DNA damage, and this phenomenon is implicated in the process of tumorigenesis. The concept of immunosenescence, postulated to manifest in elderly individuals, is defined by an age-related decline in T cells and a simultaneous elevation in proinflammatory status, leading to a diminished efficacy in response to immunotherapy. Notably, despite the rising prevalence of cancer in the elderly population, their underrepresentation in clinical trials persists. This underscores the unmet need to evaluate the safety and efficacy of cancer treatment in the elderly. This retrospective, single-center cohort study aimed to assess and evaluate the effectiveness and safety of immunotherapy in patients compared to younger individuals with metastatic solid tumors receiving ICI. A total of 220 patients were included, mostly males, with a median age of 64. The proportion of patients ≥ 65 years old was 56.5%. The use of ICI showed no significant differences concerning overall survival (OS) and progression-free survival (PFS) among age groups across different cancer types (melanoma, non-small-cell lung cancer (NSCLC), renal, and bladder cancer; p = 0.388). Concerning the response to treatment in renal cancer patients, a significant difference was observed (p = 0.041), suggesting a potential negative impact of age on the treatment response. In patients that presented immune-related adverse events (irAEs), oral corticosteroid therapy was marginally associated (p = 0.059) with the elderly population. When evaluating the NSCLC population alone (n = 131, 59.5%), our study revealed a strong association between the development of irAEs, patients' PFS and OS, and the duration of ICI treatment, but not directly correlated with age. The NSCLC elderly population presented a marginally greater number of irAEs, although without statistical significance (p = 0.86). ICI maintained efficacy and safety in elderly patients, challenging the notion that age alone should determine treatment decisions. The findings emphasize the necessity of a comprehensive geriatric assessment rather than relying solely on chronological age for personalized cancer treatment in the elderly population. Further prospective studies are needed to better understand immune responses in older adults and derive predictive biomarkers for cancer treatment.

Best Supportive Care of the Patient with Oesophageal Cancer.

BACKGROUND: Oesophageal cancer patients have poor survival, and most are unfit for curative or systemic palliative treatment. This article aims to review the best supportive care for oesophageal cancer, focusing on the management of its most frequent or distinctive symptoms and complications. METHODS: Evidence-based review on palliative supportive care of oesophageal cancer, based on Pubmed search for relevant clinical practice guidelines, reviews and original articles, with additional records collected from related articles suggestions, references and societies recommendations. RESULTS: We identified 1075 records, from which we screened 138 records that were related to oesophageal cancer supportive care, complemented with 48 additional records, finally including 60 records. This review summarizes the management of oesophageal cancer-related main problems, including dysphagia, malnutrition, pain, nausea and vomiting, fistula and bleeding. In recent years, several treatments have been developed, while optimal management is not yet standardized. CONCLUSION: This review contributes toward improving supportive care and decision making for oesophageal cancer patients, presenting updated summary recommendations for each of their main symptoms. A robust body of evidence is still lacking, and the best supportive care decisions should be individualized and shared.

Different inoculum of Leishmania braziliensis concentrations influence immunopathogenesis and clinical evolution in the ear dermis hamster model of cutaneous leishmaniasis.

The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 105 parasites. Animals infected in the ear dermis developed a disease, with an increased parasite load that more closely resembled human cutaneous leishmaniasis lesions comparing to the group infected in the footpad. Next, we evaluated if different parasite concentrations (104 , 105 and 106 ) inoculated in the ear dermis would impact the course and clinical aspects of infection. Hamsters infected with 104 and 105 parasites developed mild lesions compared to the group infected with 106 that presented severe and persistent lesions. The parasite load varied between the different parasite concentrations. The inflammatory response was more intense when infection was initiated with 106 parasites accompanied by an increased initial expression of IL-4, IL-10 and arginase in the lymph node followed by expression of both pro-and anti-inflammatory cytokines comparing to groups infected with 104 and 105 parasites. In conclusion, the number of parasites inoculated, and the initial site of infection could influence the inflammatory response, and clinical presentation. Our results suggest that the ear dermis infection model induces a chronic disease that relates to immunopathological aspects of CL natural infection.

Increased levels of cortisol are associated with the severity of experimental visceral leishmaniasis in a Leishmania (L.) infantum-hamster model.

BACKGROUND: Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. METHODS: L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. RESULTS: All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1ß, IL-10, and transforming growth-factor-ß (TGF-ß) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. CONCLUSIONS: These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity.

A Cytokine Network Balance Influences the Fate of Leishmania (Viannia) braziliensis Infection in a Cutaneous Leishmaniasis Hamster Model.

The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis. Immunopathological mechanisms are well established in the L. (L.) major-mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluated the natural history of L. braziliensis infection from its first stages up to lesion establishment, with the aim of identifying immunological parameters associated with the disease outcome and parasitism fate. To this end, hamsters infected with 104, 105, or 106 promastigotes were monitored during the first hours (4h, 24h), early (15 days, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginase gene expression were quantified in the established lesions by reverse transcription-quantitative PCR. Compared to the 105 or 106 groups, 104 animals presented lower lesions sizes, less tissue damage, and lower IgG levels. Basal gene expression in normal skin was high for TGF-ß, and intermediary for TNF, IL-6, and IL-4. At 4hpi, no cytokine induction was observed in the 104 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 106 group. At 15dpi, lesion appearance was accompanied by an increased expression of all assessed cytokines, markedly in the 105 and 106 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 104 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associated to parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi. A strong positive network correlation was observed in the 104 group, but not in the 105 or 106 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-ß are linked o L. braziliensis progression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage.

IncX4 Plasmid Carrying the New mcr-1.9 Gene Variant in a CTX-M-8-Producing Escherichia coli Isolate Recovered From Swine.

We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to ß-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus bla CTX-M-8, a bla ESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.

Molecular Epidemiology and Risk Factors of Carbapenemase-Producing Enterobacteriaceae Isolates in Portuguese Hospitals: Results From European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE).

In Portugal, the epidemiological stage for the spread of carbapenemase-producing Enterobacteriaceae (CPE) increased from sporadic isolates or single hospital clones (2010-2013), to hospital outbreaks, later. Here we report data from a 6-month study performed under the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). During the study period, 67 isolates (61 Klebsiella pneumoniae and 6 Escherichia coli) non-susceptible to carbapenems were identified in participant hospital laboratories. We detected 37 bla KPC-type (including one new variant: bla KPC-21), 1 bla GES-5, and 1 bla GES-6 plus bla KPC-3, alone or in combination with other bla genes. Bioinformatics analysis of the KPC-21-producing E. coli identified the new variant bla KPC-21 in a 12,748 bp length plasmid. The bla KPC-21 gene was harbored on a non-Tn4401 element, presenting upstream a partial ISKpn6 (ΔISKpn6/ΔtraN) with the related left IR (IRL) and downstream a truncated Tn3 transposon. PFGE and MLST analysis showed an important diversity, as isolates belonged to distinct PFGE and STs profiles. In this study, we highlighted the presence of the high-risk clone E. coli sequence-type (ST) 131 clade C/H30. This worldwide disseminated E. coli lineage was already detected in Portugal among other antibiotic resistance reservoirs. This study highlights the intra- and inter-hospital spread and possible intercontinental circulation of CPE isolates.

Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis.

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions. METHODS: Hamsters were intradermally infected in the footpad with 10(5) promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-É£, IL-10, TGF-ß TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology. RESULTS: A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-É£, TGF-ß, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-ß to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-ß to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-É£, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7-1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant. CONCLUSIONS: In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.

Intranasal vaccination with leishmanial antigens protects golden hamsters (Mesocricetus auratus) against Leishmania (Viannia) Braziliensis infection.

BACKGROUND: Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania) amazonensis antigens (LaAg) partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia) braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection. METHODOLOGY/PRINCIPAL FINDINGS: Golden hamsters vaccinated with either two intranasal (IN) doses of LaAg (10 µg) or two intramuscular doses of LaAg (20 µg) were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM) immunisation failed to provide protection. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.

Comparative evaluation of lesion development, tissue damage, and cytokine expression in golden hamsters (Mesocricetus auratus) infected by inocula with different Leishmania (Viannia) braziliensis concentrations.

The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.

Golden hamster (Mesocricetus auratus) as an experimental model for Leishmania (Viannia) braziliensis infection.

The lack of an adequate model for Leishmania (Viannia) braziliensis infection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropic Leishmania infection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, different Leishmania isolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility of L. braziliensis infection in this model. The natural history of L. braziliensis infection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-Leishmania IgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused by L. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

Different isolates from Leishmania braziliensis complex induce distinct histopathological features in a murine model of infection.

The aim of this study was to evaluate the histopathological features in tissues of mice infected by human isolates (I, II, and III) or the reference M2903 strain of Leishmania braziliensis complex. BALB/c and C57Bl/6 mice were infected in the hind footpad with 10(6) stationary-phase promastigotes of L. braziliensis complex. The evolution of lesions was observed for 10 weeks and the animals were then euthanized and liver, spleen and popliteal lymph nodes were collected. Tissues were stained with hematoxylin and eosin and analyzed by immunohistochemistry assay. Increased thickness of infected footpads was observed in all animals, lesions were nodular and non-ulcerated. Mice infected with isolate I presented inflammatory infiltrates consisting predominantly of mononuclear cells in all tissues examined, and also a great number of megakaryocytes, compared with other isolates. Infection with isolate II led to an infected footpad enlargement not seen in other isolates. In addition, mononuclear infiltrates in the liver and hemosiderin in spleen were noted. Conversely, mice infected with either isolate III or M2903 strain only showed an increased number of megakaryocytes in spleen. All tissues examined had detectable amastigote forms of Leishmania by immunohistochemistry in all groups. Taking together, our results showed an unforeseen behavior of different isolates of L. braziliensis complex that led to diverse pathological findings.