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Background: Female breast cancer (BC) remains the most common cause of total cancer deaths around the world. Several studies have investigated BC biomarkers, but vital circulating biomarkers for early diagnosis of malignancy are still scarce. Thus, finding sensitive, selective and accurate biomarkers is required to get better BC outcome and to prolong patients’ survival. Therefore, this review investigated the feasibility of using circulating trace elements (TEs) as the new promising biological biomarkers for BC diagnosis and prevention. Methods: We systematically searched EMBASE, Medline, Google Scholar, PubMed, SciELO, Scopus databases or Web of Science for orginal studies presenting the significant changes in the concentrations of circulating TEs in terms of serum, plasma or blood from female breast cancer patients. Results: The search yielded 2697 articles, of which 39 were considered for this review. The study showed that four essential TEs (Se, Cu, Zn and Mn) significantly decreased when only one essential trace elemnt (Fe) increased consistently, while five toxics circulating TEs (Cd, Cr, Pb, Co, Mo) increased significantly with a significant difference compared to healthy groups. The essential TEs, Se and Cu were reported to decrease the most in fifteen and twenty-one studies, respectively. However, regarding the toxic circulating TEs, Cd and Pb were found to increase most significantly in seven studies. Among the essential TEs, Se and Zn were reported to have the most potential, with Cd and Pb having the most potential for use as new promising biomarkers to diagnose or prevent BC. Conclusion: The findings provide an insight into the TEs circulating biomarkers for early BC diagnosis and prevention. Due to its high heterogeneity, meta-analysis was not assessed; hence, futher investigation may be required on their clinical outcomes in BC with high sensitivity and specificity for accurate therapeutic response.

The renin angiotensin system (RAS) is composed of several enzymes and substrates on which angiotensin converting enzyme (ACE) 1 and renin act to produce angiotensin II. ACE1 and its substrates control blood pressure, affect cardiovascular and renal function, hematopoiesis, reproduction, and immunity. The increased expression of ACE1 has been observed in human monocytes during congestive heart failure and abdominal aortic aneurysm. Moreover, T lymphocytes from hypertensive patients presented increased expression of ACE1 gene after in vitro stimulation with Angiotensin-II (AngII) with the highest ACE1 expression observed in hypertensive patients with low-grade inflammation. Our group and others have shown that aging is associated with comorbidities, chronic inflammation and immunosenescence, but there is a lack of data about ACE1 expression on immune cells during the aging process. Therefore, our aim was to evaluate the levels of ACE1 expression in non-lymphoid as compared to lymphoid cells in association with the immunosenescence profile in adults older than 60 years. Cryopreserved peripheral blood mononuclear cells obtained from blood samples were used. Cells were stained with monoclonal antibodies and evaluated by flow cytometry. We found that ACE1 was expressed in 56.9% of non-lymphocytes and in more than 90% of lymphocytes (all phenotypes). All donors exhibited characteristics of immunosenescence, as evaluated by low frequencies of Naive CD4+and CD8+T cells, high frequencies of EMRA CD8+T cells, and double-negative memory B cells. These findings in addition to the increased C-reactive protein levels are intriguing questions for the study of ACE1, inflammaging, immunosenescence and perspectives for drug development or repurposing.

Since 1990 it has been observed a significant increase of the global aged population with a perspective of 1.5 billion of older individuals by 2050 and 3 billion by 2100 due to increased longevity. To guarantee healthy longevity to this population, public policies focused in economic, social and health aspects are crucial. Healthy longevity is influenced by genetics, habits (i.e. healthy nutrition, physical activity, non-smoking) and environment. Several changes occur in organs and tissues during the ageing process, therefore the immune system also suffers alterations that could play a role in age-related conditions (cardiovascular, respiratory, neurologic, tumoral, and infectious). Recently we had a clear example of how an infectious disease (COVID-19) is exacerbated in aged patients with high morbidity and mortality (mainly in those with comorbidities). It was found an association between elevated morbidity/mortality after SARS-CoV-2 and increased expression of inflammageing and immunosenescence markers. To fight immunosenescence it has been proposed that healthy nutrition and supplements (vitamins B, D, C, or E, essential minerals, and amino acids) could be used. In addition, physical activity has been suggested for the maintenance of adequate function of respiratory and cardiovascular systems, memory, and immune system. Considering the complexity of healthy longevity, this introduction will briefly comment some alterations occurring in the innate and adaptive immunity during the ageing process and possible alternatives of intervention in order to reach old age with functionality. The complete discussion about these changes will be distributed in the Chaps. 2– 9.

Secondary autoimmune diseases (2ndADs), most frequently autoimmune cytopenias (AICs), were first described after allogeneic hematopoietic stem cell transplantation (HSCT) undertaken for malignant and hematological indications, occurred at a prevalence of ~5-6.5%, and were attributed to allogeneic immune imbalances in the context of graft versus host disease, viral infections, and chronic immunosuppression. Subsequently, 2ndADs were reported to complicate roughly 2-14% of autologous HSCTs performed for an autoimmune disease. Alemtuzumab in the conditioning regimen has been identified as a risk for development of 2ndADs after either allogeneic or autologous HSCT and is consistent with the high rates of 2ndADs when using alemtuzumab as monotherapy. Due to the significant consequences but variable incidence, depending on conditioning regimen, of 2ndADs and similarity in known immune reconstitution kinetics after autologous HSCT for autoimmune diseases and after alemtuzumab monotherapy, we propose that an imbalance between B and T lineage regeneration early after HSCT may underlie the pathogenesis of 2ndADs.

The challenge of distinguishing between changes attributable to ageing and those attributable to pathology is even greater for the immune system than for many other organs, and this is especially true for myeloid-derived suppressor cells (MDSCs). Hematopoiesis is different in older adults with a bias towards myelopoiesis, and older adults also manifest "inflammageing" exacerbated by disease and contributing to MDSC induction. Hence, at least in humans, one can only investigate MDSCs in the context of ageing and disease states, and not in the context of ageing processes per se. This contribution provides a brief overview of the literature on MDSCs and ageing in humans.

OBJECTIVES: Myeloid-derived suppressor cells contribute to the immunosuppressive microenvironment during tumor development and limit the efficacy of cancer immunotherapy. Identifying myeloid-derived suppressor cells and associated factors is the first step in creating strategies to reverse the suppressive effects of these cells on the immune system. METHODS: To induce lung cancer, we administered 2 doses of urethane to BALB/c mice and observed these animals for 120 days. After this period, we evaluated the percentage of myeloid-derived suppressor cells in the blood, lung and bone marrow. The expression of alpha-smooth muscle actin, transforming growth factor-β, Toll-like receptor 2, Toll-like receptor 4, and interleukin-6 was also determined in the lung tissue. RESULTS: Myeloid-derived suppressor cells were increased in all evaluated tissues after lung cancer development in association with increased Toll-like receptor 4 expression and decreased interleukin-6 expression in the lung. We observed alpha-smooth muscle actin and transforming growth factor-β expression in lung nodules. CONCLUSIONS: We believe that the early diagnosis of cancer through determining the blood levels of myeloid-derived suppressor cells followed by the depletion of these cells should be further investigated as a possible approach for cancer treatment.

Worldwide the percentage of individuals older than 60 years is growing due to the increase in longevity. Age is an important risk factor for cancer and subjects aged over 60 also have a higher risk of comorbidities. Approximately 50 % of neoplasms occur in patients older than 70 years and these patients often have a range of therapies ranging from surgery, adjuvant or neoadjuvant therapy, or even palliative chemotherapy. While the OPAS (Pan American Health Organization) considers elderly subjects to be those aged over 60 years and for the WHO (World Health Organization) it is subjects over 65 years, a major concern for poor prognosis is with cancer patients over 70–75 years. These patients have a lower functional reserve, a higher risk of toxicity after chemotherapy, and an increased risk of infection and renal complications that lead to a poor quality of life. Several biomarkers (immunological and physiological) have been evaluated in cancer aiming to improve diagnostics, to predict whether a patient could have any benefit from a specific treatment, and to perform patient follow-up. Cancer is a complex disease and in this chapter we will discuss the distribution of cancer cases in older adults, the most important types of cancer in this population, treatment and outcomes, and possible immunosenescence characteristics related to cancer in the aged.

Spleen or spleen plus bone marrow cells from (BALB/c × C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4 + and CD8 + T cells 7 and 21 days after donor cell transfer. The populations of CD8 + CD45RB low and CD8 + CD44 high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4 + T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8 + T cells leading to enhanced graft survival.

Calcineurin inhibitors (CNIs) are routinely used in immunosuppressive therapy and both Cyclosporine (CsA) and Tacrolimus (FK506) show similar efficacies to prevent rejection and death within the first year after organ transplantation. However, their use is limited by side effects such as kidney damage, hypertension, onset of diabetes and hyperlipidemia. It is a consensus that compared with CsA, FK506 causes less changes in blood pressures, serum lipids and renal function. Nevertheless, FK506 use is associated with a higher incidence of post-transplant diabetes mellitus (PTDM). FTY720 is a new compound that has shown a protective effect in animal models with respect to rejection in transplantation, ischemia–reperfusion injury, autoimmune diseases and tumor development. FTY720 acts by altering lymphocytes homing from blood to peripheral lymphoid organs. In mice, FTY720 administered in combination with CsA during 21 days has prolonged skin allograft survival without causing significant renal changes. In a model of CsA-induced chronic nephropathy in rats, FTY720 administration prevented renal injury suggesting benefit from using a combination of these drugs. In a canine kidney allograft model, FTY720 in combination with low doses of CsA or FK506 showed an addictive anti-rejection effect without causing critical adverse effects. We therefore, investigated whether 21 days of FTY720 administration in association with FK506 could prevent renal damage and development of diabetes in mice. Mice receiving FK506 alone or FTY720 + FK506 during 21 days showed changes in kidney function and structure besides an increase in blood glucose and lymphopenia. The FTY720 + FK506 combination requires further investigation with an aim toward understanding the mechanisms involved with respect to side effects.

Aging has been associated with progressive molecular and structural changes causing loss of function in several organs. There is a general hypothesis that very old individuals suffer profound modifications where different domains (immune, metabolic and cognitive) loose the tight functional interconnection present in younger bodies. However, it is not clear how this interconnection is affected at the very early stage of ageing and whether gender plays a role. Therefore, our aim was to evaluate some biological markers in non-institutionalized “healthy individuals” at the very early stage of aging (60 to 65 years old, female and male). Blood was collected and serum creatinine, albumin and glucose were measured. In addition, we evaluated these individuals for lymphocytes phenotype (T CD4+, T CD8+, CD19+) by flow cytometry in peripheral mononuclear blood cells. It was observed that at the early stage of ageing male present higher creatinine and albumin serum levels compared to female. In addition, male had higher percentages of effector memory CD4+ and CD8+ T cells and lower percentages of naive CD8+ T cells. No differences were observed for B cells. These findings suggest that metabolic functions and immune system are compromised in male individuals at the very early stage of ageing and thus gender differences should be considered for the design of new therapies to the elderly.

Infections, cancer and autoimmune diseases occur more frequently in the elderly, and although many factors contribute to this, the age-related remodelling of the immune system, termed immunosenescence, plays a major role. Over the last two decades, studies have evaluated the effect of ageing on both the adaptive and innate arms of the immune system and demonstrated compromised function in several cells including lymphocytes (naïve, effector and memory), regulatory T and B cells, monocytes, neutrophils and NK cells. In addition, a well-documented feature of ageing is the increase in systemic inflammatory status (inflammageing), with raised serum levels of IL6, TNFα and CRP as well as reduced IL10. Recently, myeloid-derived suppressor cells have been the focus of many reports as these cells show immunosuppressive properties and are present in higher frequency during infections, cancer and autoimmunity. Importantly, there have been publications showing increased numbers of myeloid-derived suppressor cells in aged mice and humans. In this review, we discuss the current literature on myeloid-derived suppressor cells, their possible role in altered immune function in the elderly, and whether it may be possible to manipulate these cells to alleviate age-related immune dysfunction.

The skin is comprised of the epidermis and its adnexa, the dermis, and the hypodermis, resting on the subcutaneous tissue which is essentially adipose tissue (Fig. 2.1).

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.

OBJECTIVES: Both renal function and immune system function decline with age. Although controversial, a significant number of studies have shown that the decline in kidney function is associated with the worsening of the immune system. These findings are reinforced by the increased susceptibility to infections and deficient immunization coverage after vaccination both in patients with chronic renal disease and in elderly individuals. Our objective was to evaluate a non-institutionalized elderly population from São Paulo City and correlate the estimated glomerular filtration rate with the percentage of lymphocytes in circulation. METHODS: A random population of 237 individuals (107 men and 130 women), ranging in age from 60 to 101 years, who were enrolled in the Health, Well-Being and Aging Study was evaluated for renal function (Modification on Diet in Renal Disease formula) and lymphocyte percentage (flow cytometry). RESULTS: Aging was associated with a decrease in the estimated glomerular filtration rate in both male and female individuals. We did not identify a significant correlation between the estimated glomerular filtration rate and either the percentage of CD4, CD8, and B cells or CD4/CD8 ratio. The median percentage of CD8+ T cells was significantly lower in individuals with an estimated glomerular filtration rate ≥60 mL/min/1.73 m2. CONCLUSIONS: In this study, no statistical correlation was found between the estimated glomerular filtration rate and either the lymphocyte phenotype (CD4+, CD8+, and CD19+ cells) or the CD4/CD8 ratio in blood.

OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) BACKGROUND: Age-associated changes in the immune system cause decreased protection after vaccination and increased rates of infections and tumor development. METHODS: Lymphocyte percentages were compared by gender and age to establish differences between subtypes. Three mL blood samples were obtained from 218 randomly selected individuals (60-101 years old) who live in São Paulo city. Blood was lysed with Tris phosphate buffer and stained for 30 minutes with monoclonal antibodies (CD3PerCP, CD4FITC, CD8Pe, CD19Pe) for analysis by flow cytometry. Statistical analysis was by ANOVA. RESULTS: The percentage of CD4+ T cells (p-value = 0.005) and the CD4/CD8 ratio (p-value = 0.010) were lower in men, whereas the percentage of CD8+ T cells was lower (p-value = 0.002) in women; the percentage of B cells (CD19+ ) was similar between groups. Individuals grouped by gender and age range and compared showed a drop in CD4+ cells in 75 to 79-year-old men (female: 46.1% ± 8.1% and male: 38.8% ± 10.5%; p-value = 0.023). Also, the 80 to 84-year-old group of men had a higher percentage of CD8+ (female: 20.8% ± 8.2%, and male: 27.2% ± 8.2%; p-value = 0.032). Low percentages of B cells were detected in men in the 75 to 79-year-old (p-value = 0.003), 85 to 89-year-old (p-value = 0.020) and older than 90 year old (p-value = 0.002) age ranges. CONCLUSION: Elderly men present with more changes in lymphocyte subsets compared to elderly women. These findings could demonstrate impairment in the immune response since the lower CD4+ in men would provide less help to B cells (also lower in men) in terms of antibody production. In addition, the increase in CD8+ cells in this group could represent chronic inflammation observed during the aging process. Universidade Federal de São Paulo (UNIFESP) Immunology Department Universidade de São Paulo Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Escola de Saúde Pública Universidade Federal de São Paulo (UNIFESP) Biosciences Department UNIFESP, Immunology Department UNIFESP, Biosciences Department SciELO

Acute kidney injury (AKI) is characterized by a relatively sudden decrease in the production, processing, and excretion of ultrafiltrate by the kidney (decrease in glomerular filtration rate – GFR). Acute kidney injury (AKI) caused by ischemia and reperfusion injury (IRI) is a common event in transplantation and 20% to 80% of kidneys from deceased donors can present delayed graft function (DGF) depending on the injury extent (Perico et al., 2004). After transplantation it could be expected immediate renal function, slow recovery function, non-oliguric acute tubular necrosis (ATN), total anuria. Delayed graft function (DGF) is defined by transplant centers as: the need of dialysis (at least one session) during the first week post-transplantation (Koning et al., 1997), early urine output lower than 1200mL/day or no decrease in serum creatinine within 48h (Shoskes et al., 2001), creatinine clearance lower than 10mL/min (Giral-Classe et al., 1998), creatinine at day 10 higher than 221μmol/L (Cosio et al., 1997). Delayed graft function has been considered an independent predictor of graft loss since multivariate analysis showed a relative risk of graft loss 2.9 times greater for DGF than for kidneys with immediate function (Halloran et al., 1988). The US Renal Data System (37,000 primary cadaver transplants) showed a relative risk of 1.53 for 5-year graft loss in association with DGF (Ojo et al., 1997). In cadaver transplants (1994-1998 in USA) the halflife of kidneys with DGF was 7.2 years whereas in kidneys with immediate function it was 11.5 years (Halloran et al., 2001). In the presence of rejection DGF’s effect is even stronger and kidney graft half-life decreases from 9.4 to 6.2 years (Shoskes et al., 1998). Kyllonen et al. (2000) showed in a follow-up of 1047 cadaveric kidney transplants performed at University of Helsinki that 5-years graft survival was 60% in patients presenting DGF and rejection, 73% in patients with rejection, 77% in patients with DGF and 88% in patients without both risk factors. They concluded that DGF was a significant factor affecting long-term graft survival, both through and independent of acute rejection. In 10-years of transplantation follow-up Troppman et al. (1999) observed 64% of graft survival in patients without DGF or rejection episodes, 44% in patients with DGF, 36% in patients with rejection, and 15% in patients presenting both risk factors. A range of factors could lead to DGF such as organ procurement (i.e. kidneys from nonheart-beating donors), donor characteristics (i.e. donors older than 55 years), period of ischemia, recipient historic (i.e. number of recipient’s previous transplants), renal toxicity, ureteral obstruction, among others. Since DGF is considered an independent risk for graft

Tumor growth occurs by the imbalance between cells with effector function and cells with suppressor/regulatory functions. To investigate this scenario we administered the chemical carcinogen Urethane in BALB/c mice and followed these animals during 120 days to observe lung tumor development. In another set of experiments the same protocol was performed with the harvest of spleen, lung and blood at 20 and 30 days after Urethane injection. The lung was used for histology, spleen cells were evaluated for IFN-γ production, and serum nitrite was measured as an indirect form of nitric oxide (NO) evaluation. The spleen and lung-infiltrating cells were evaluated by flow cytometry for CD11b+Gr-1+ myeloid suppressor cells and CD4+FoxP3+ T regulatory cells. Urethane led to lung nodules development after 120 days and the time point evaluation showed that splenocytes stimulated ex vivo expressed higher levels of IFN-γ 20 days after the chemical injection. Also, the level of nitric oxide in serum was higher after 20 days of Urethane injection. There was no statistical difference in spleen cells percentages for CD11b+Gr-1+ and CD4+Foxp3+ in all groups. However, lung-infiltrating cells presented early (20 days) a higher expression of CD11b+Gr-1+ suggesting suppression at this site. In conclusion, it was possible to observe two distinct events at the very early time point after Urethane injection. In periphery there was an increase at the effector immune response (as depicted by IFN-γ-producing cells) and in tumor development site there was an increase at the suppressor cell (CD11b+Gr-1+) phenotype. Suppressor/regulatory cells are targets for cancer therapy.

Uveitis is an intraocular inflammatory disease causing a significant visual impairment. The disease can be idiopathic, associated with infectious and systemic disorders or arisen from an unknown cause. Over the last 20 years the model of EAU in mice has contributed significantly for the establishment of parameters for diagnostic evaluations and therapies for posterior uveitis in human. Many studies using recently discovered molecules which present proinflammatory and anti-inflammatory effects have been described. Moreover, new approaches of research provided by the increasing body of knowledge on components of immune responses such as cytokines, T-cell subpopulations and their associated functions have contributed for the further understanding of uveitis and possible treatment.