Outcome of Second Primary Malignancies Developing in Multiple Myeloma Patients.

BACKGROUND: There is an increased risk of second primary malignancies (SMPs) in patients with multiple myeloma (MM). This multinational 'real-world' retrospective study analyzed the characteristics and outcomes of MM patients that developed SPMs. RESULTS: 165 patients were analyzed: 62.4% males; 8.5% with a prior cancer; 113 with solid SPMs, mainly ≥stage 2; and 52 with hematological SPM (hemato-SPM), mainly MDS/AML. Patients with hemato-SPM were younger (p = 0.05) and more frequently had a prior AutoHCT (p = 0.012). The time to SPM was shorter in the older (>65 years) and more heavily pretreated patients. One hundred patients were actively treated at the time of SPM detection. Treatment was discontinued in 52, substituted with another anti-MM therapy in 15, and continued in 33 patients. Treatment discontinuation was predominant in the patients diagnosed with hemato-SPM (76%). The median OS following SPM detection was 8.5 months, and the main cause of death was SPM. A poor ECOG status predicted a shorter OS (PS 3 vs. 0, HR = 5.74, 2.32-14.21, p < 0.001), whereas a normal hemoglobin level (HR = 0.43, 0.19-0.95, p = 0.037) predicted longer OS. CONCLUSIONS: With the continuing improvement in OS, a higher proportion of MM patients might develop SPM. The OS following SPM diagnosis is poor; hence, frequent surveillance and early detection are imperative to improve outcomes.

Assessing the frequency of CD163+ tumor-associated macrophages and CD3+ T lymphocytes between MGUS and plasma cell myeloma.

Plasma cell dyscrasias (PCDs) are a heterogeneous group of diseases, and the most common is monoclonal gammopathy of undetermined significance (MGUS). This premalignant PCD consistently precedes multiple myeloma (MM), with a 1% risk of progression per year. Evading and suppressing the host immune system is an important step in the progression of MGUS to MM. This pilot study was designed to assess whether MGUS and MM have a distinct microenvironment, characterized by a unique distribution of immune cells, including tumor-associated macrophages. Evaluation of bone marrow (BM) tumor microenvironment was performed using immunohistochemical quantification of T cells (CD3), macrophages (CD68), and a macrophage subtype (CD163). The findings were compared between MGUS and MM to determine whether differences existed. The results suggest that there is a significantly lower percentage of CD3-positive, CD68-positive and CD163-positive immune effector cells in BM trephine biopsy samples from patients with MGUS than in those from patients with untreated MM (p < 0.001). Interestingly, in a patient treated for MM, the percentages of CD3+ and CD68+ cells were the same as those in other patients with untreated MM; however, the percentage of CD163+ cells reduced and correlated with low plasma cell count. Future studies are required to investigate whether the percentage of CD163+ cells is correlated with disease burden in patients with MM. If this is the case, then the level of soluble CD163 in plasma could be a potential biomarker of disease burden in patients with nonsecretory myelomas, in whom measurements of levels of paraprotein and free light chains are inconclusive.

CD169+ Monocyte and Regulatory T Cell Subsets Are Associated with Disease Activity in Rheumatoid Arthritis.

Disease activity in rheumatoid arthritis (RA) is influenced by activation of circulating and synovial immune cells. Regulatory T cells (Tregs) and monocytes are key cells that drive inflammation in RA. This study investigated if a relationship exists between disease activity in RA and circulating Treg and monocyte numbers and phenotypes. A potential sialic acid (Sia) mediated link between Tregs and monocytes was also probed in vitro. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patient (n = 62) and healthy control (n = 21) blood using density gradient separation. Flow cytometry was used to count and phenotype Treg and monocyte subsets, and to sort healthy control Tregs for Sia cell culture experiments. The effects of Sia on activated Treg FoxP3 and NFκB expression was assessed by flow cytometry and concentrations of secreted TNFα, IL-10 and IFNγ determined by ELISA. High disease activity RA patients who were unresponsive to disease modifying anti-rheumatic drugs (n = 31), have significantly lower relative numbers (percentages) of CD4+CD25+CD127− Tregs (p < 0.01) and memory CD45RA−FoxP3+ Tregs (p < 0.01), compared to low disease activity responders (n = 24). Relative numbers of non-classical CD169+ monocytes are associated with disease activity in RA (p = 0.012). Sia reduced Treg expression of FoxP3, NFκB and cytokines in vitro. A strong association has been identified between non-classical CD169+ monocytes and post-treatment disease activity in RA. This study also indicates that Sia can reduce Treg activation and cytokine release. We postulate that such a reduction could be mediated by interaction with sialyted proteins captured by CD169+ monocytes.

IgD multiple myeloma: biology, diagnosis, and treatment.

IgD multiple myeloma is uncommon. Patients generally present at a younger age and have shorter progression free and overall survivals (OSs). Its rarity has inhibited development of a specific risk stratification system or informed best treatment protocols. We present interphase fluorescence in situ hybridization results from a group of 29 cases. These showed evidence of a decreased male to female ratio, decreased OS in patients aged 70 and over, better outcomes in those with kappa light chain restriction, and CD56 positive patients had longer survivals than those lacking CD56. We discuss the biology of IgD multiple myeloma, the need for prospective studies, and challenges for improvements in diagnosis and treatment. We suggest an International Register to accelerate development of best practice guidelines for diagnosis, risk stratification, and treatment.

Triple jeopardy in ageing: COVID-19, co-morbidities and inflamm-ageing.

Covid-19 endangers lives, has disrupted normal life, changed the way medicine is practised and is likely to alter our world for the foreseeable future. Almost two years on since the presumptive first diagnosis of COVID-19 in China, more than two hundred and fifty million cases have been confirmed and more than five million people have died globally, with the figures rising daily. One of the most striking aspects of COVID-19 illness is the marked difference in individuals' experiences of the disease. Some, most often younger groups, are asymptomatic, whereas others become severely ill with acute respiratory distress syndrome (ARDS), pneumonia or proceed to fatal organ disease. The highest death rates are in the older and oldest age groups and in people with co-morbidities such as diabetes, heart disease and obesity. Three major questions seem important to consider. What do we understand about changes in the immune system that might contribute to the older person's risk of developing severe COVID-19? What factors contribute to the higher morbidity and mortality in older people with COVID-19? How could immunocompetence in the older and the frailest individuals and populations be supported and enhanced to give protection from serious COVID-19 illness?

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing.

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.

Multiple myeloma with central nervous system relapse.

Central nervous system involvement in multiple myeloma is a rare complication but carries a very poor prognosis. We provide a review of current literature, including presentation, treatment and survival data, and describe our experience in a regional hematologic malignancy diagnosis center where, over a 15-year period, ten cases were identified. Although the median age of onset, frequently between 50-60 years, is comparatively young, those diagnosed usually have a preceding diagnosis of multiple myeloma and often have had several lines of treatment. We discuss putative underlying factors such as prior treatment and associations including possible risk factors and features suggestive of a distinct biology. Central nervous system involvement may be challenging to diagnose in myeloma, displaying heterogeneous symptoms that can be confounded by neurological symptoms caused by the typical features of myeloma or treatment side-effects. We discuss the clinical features, imaging and laboratory methods used in diagnosis, and highlight the importance of considering this rare complication when neurological symptoms occur at presentation or, more commonly, during the disease pathway. In the absence of clinical trial data to inform an evidence-based approach to treatment, we discuss current and novel treatment options. Finally, we propose the establishment of an International Registry of such cases as the best way to collect and subsequently disseminate presentation, diagnostic and treatment outcome data on this rare complication of multiple myeloma.

Age and Age-Related Diseases: Role of Inflammation Triggers and Cytokines.

Cytokine dysregulation is believed to play a key role in the remodeling of the immune system at older age, with evidence pointing to an inability to fine-control systemic inflammation, which seems to be a marker of unsuccessful aging. This reshaping of cytokine expression pattern, with a progressive tendency toward a pro-inflammatory phenotype has been called "inflamm-aging." Despite research there is no clear understanding about the causes of "inflamm-aging" that underpin most major age-related diseases, including atherosclerosis, diabetes, Alzheimer's disease, rheumatoid arthritis, cancer, and aging itself. While inflammation is part of the normal repair response for healing, and essential in keeping us safe from bacterial and viral infections and noxious environmental agents, not all inflammation is good. When inflammation becomes prolonged and persists, it can become damaging and destructive. Several common molecular pathways have been identified that are associated with both aging and low-grade inflammation. The age-related change in redox balance, the increase in age-related senescent cells, the senescence-associated secretory phenotype (SASP) and the decline in effective autophagy that can trigger the inflammasome, suggest that it may be possible to delay age-related diseases and aging itself by suppressing pro-inflammatory molecular mechanisms or improving the timely resolution of inflammation. Conversely there may be learning from molecular or genetic pathways from long-lived cohorts who exemplify good quality aging. Here, we will discuss some of the current ideas and highlight molecular pathways that appear to contribute to the immune imbalance and the cytokine dysregulation, which is associated with "inflammageing" or parainflammation. Evidence of these findings will be drawn from research in cardiovascular disease, cancer, neurological inflammation and rheumatoid arthritis.

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD.

Towards Stratified Medicine in Plasma Cell Myeloma.

Plasma cell myeloma is a clinically heterogeneous malignancy accounting for approximately one to 2% of newly diagnosed cases of cancer worldwide. Treatment options, in addition to long-established cytotoxic drugs, include autologous stem cell transplant, immune modulators, proteasome inhibitors and monoclonal antibodies, plus further targeted therapies currently in clinical trials. Whilst treatment decisions are mostly based on a patient's age, fitness, including the presence of co-morbidities, and tumour burden, significant scope exists for better risk stratification, sub-classification of disease, and predictors of response to specific therapies. Clinical staging, recurring acquired cytogenetic aberrations, and serum biomarkers such as ß-2 microglobulin, and free light chains are in widespread use but often fail to predict the disease progression or inform treatment decision making. Recent scientific advances have provided considerable insight into the biology of myeloma. For example, gene expression profiling is already making a contribution to enhanced understanding of the biology of the disease whilst Next Generation Sequencing has revealed great genomic complexity and heterogeneity. Pathways involved in the oncogenesis, proliferation of the tumour and its resistance to apoptosis are being unravelled. Furthermore, knowledge of the tumour cell surface and its interactions with bystander cells and the bone marrow stroma enhance this understanding and provide novel targets for cell and antibody-based therapies. This review will discuss the development in understanding of the biology of the tumour cell and its environment in the bone marrow, the implementation of new therapeutic options contributing to significantly improved outcomes, and the progression towards more personalised medicine in this disorder.

Genes and life-style factors in BELFAST nonagenarians: Nature, Nurture and Narrative.

Understanding how to 'Age Longer and Age Well' is a priority for people personally, for populations globally and for government policy. Nonagenarians are the oldest members of our societies and survivors of their generation. Approximately 10 % of nonagenarians reach 90 years and beyond in good condition and seem to have a combination of both age-span and health-span. But what are the factors which help people reach their ninetieth birthday and beyond in good condition? Are they genetics, as in 'nature', or do they depend on 'nurture' and are related to environment, or are both factors inextricably intertwined within the concept of behavioural genetics? Nonagenarians have rich life experiences that can teach us much about ageing well; they are reservoirs of genetic, life-style and behavioural information which can help dissect out how to live not only longer but better. Personal family history and narrative are powerful tools that help to determine familial traits, beliefs and social behaviours and when used in parallel with new biotechnology methods inform and elaborate causality. Here we present themes and insights from personal narrative enquiry from nonagenarian participants from the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST) about factors they consider important for good quality ageing and relate these insights to the emerging genetics and life-style evidence associated with healthy longevity.

Killer Immunoglobulin-like Receptors (KIR) haplogroups A and B track with Natural Killer Cells and Cytokine Profile in Aged Subjects: Observations from Octo/Nonagenarians in the Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST).

BACKGROUND: Natural Killer Cells (NK) play an important role in detection and elimination of virus-infected, damaged or cancer cells. NK cell function is guided by expression of Killer Immunoglobulin-like Receptors (KIRs) and contributed to by the cytokine milieu. KIR molecules are grouped on NK cells into stimulatory and inhibitory KIR haplotypes A and B, through which NKs sense and tolerate HLA self-antigens or up-regulate the NK-cytotoxic response to cells with altered HLA self-antigens, damaged by viruses or tumours. We have previously described increased numbers of NK and NK-related subsets in association with sIL-2R cytokine serum levels in BELFAST octo/nonagenarians. We hypothesised that changes in KIR A and B haplotype gene frequencies could explain the increased cytokine profiles and NK compartments previously described in Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST) octo/nonagenarians, who show evidence of ageing well. RESULTS: In the BELFAST study, 24% of octo/nonagenarians carried the KIR A haplotype and 76% KIR B haplotype with no differences for KIR A haplogroup frequency between male or female subjects (23% v 24%; p=0.88) or for KIR B haplogroup (77% v 76%; p=0.99). Octo/nonagenarian KIR A haplotype carriers showed increased NK numbers and percentage compared to Group B KIR subjects (p=0.003; p=0.016 respectively). There were no KIR A/ B haplogroup-associated changes for related CD57+CD8 (high or low) subsets. Using logistic regression, KIR B carriers were predicted to have higher IL-12 cytokine levels compared to KIR A carriers by about 3% (OR 1.03, confidence limits CI 0.99-1.09; p=0.027) and 14% higher levels for TGF-ß (active), a cytokine with an anti-inflammatory role, (OR 1.14, confidence limits CI 0.99-1.09; p=0.002). CONCLUSION: In this observational study, BELFAST octo/nonagenarians carrying KIR A haplotype showed higher NK cell numbers and percentage compared to KIR B carriers. Conversely, KIR B haplotype carriers, with genes encoding for activating KIRs, showed a tendency for higher serum pro-inflammatory cytokines compared to KIR A carriers. While the findings in this study should be considered exploratory they may serve to stimulate debate about the immune signatures of those who appear to age slowly and who represent a model for good quality survivor-hood.

Intracellular cytokine production and cognition in healthy older adults.

Elevated concentrations of the pro-inflammatory cytokines IL-1ß and IL-6 have been associated with impaired cognitive performance. There are, however, few studies that have examined the relationship between cytokine production and specific aspects of cognition in healthy older individuals. Two-colour flow cytometry was used to determine intracellular cytokine production by activated monocytes, and neuropsychological tests were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) in 93 apparently healthy men and women aged 55-70 years. A series of hierarchical regression analyses was carried out to examine the contribution of IL-1ß and IL-6 (% expression and production (antibody binding capacity (ABC))) to recognition, attention and working memory, after controlling for socio-demographic variables (age, sex and social class). IL-1ß% expression and IL-6 production predicted aspects of working memory. Recognition memory was found to be sensitive to the affects of age and social class. The current study suggests that higher intracellular cytokine production by activated monocytes may be predictive of lower cognitive performance in working memory in healthy older individuals. These findings indicate that utilization of models for in vivo cytokine production upon immune challenge may be useful in studying specific aspects of memory affected during inflammatory responses, for example in individuals at risk for cognitive decline owing to age-related inflammatory disorders.

Neither P-gp SNP variants, P-gp expression nor functional P-gp activity predicts MDR in a preliminary study of plasma cell myeloma.

INTRODUCTION: Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) can compromise the successful treatment of many malignancies including plasma cell myeloma (PCM). However, methods do not yet exist that can accurately determine P-gp activity in PCM patient samples. METHODS: In this study, we have utilized new advances in flow cytometric methods to determine the activity of P-gp in PCM tumor cells. Furthermore, we have used several PCR-based approaches to perform a pilot study determining the functional impact of ABCB1 SNPs in patients with PCM. RESULTS: No associations were seen between P-gp activity or expression and subgroups of PCM. Similarly, no association was seen between P-gp expression and SNPs within ABCB1 although a nonsignificant reduction in activity was demonstrated for rs1045642 (P = 0.121). CONCLUSIONS: We have described a new method for the determination of P-gp and MRP activity suitable for use in clinical studies and have optimized this method to include a gating strategy, allowing routine use on PCM bone marrow aspirate samples. This is the first patient study to consider the full impact of SNPs within ABCB1 all the way from the genome to the proteome in PCM. The methods described here could also be utilized for future studies of "stem cell like" side populations in PCM that are considered to be drug resistant. Furthermore, minor amendments to these methods will facilitate studies of P-gp, MRP, and BCRP activity in other haematological malignancies.

Multidrug resistance gene expression and ABCB1 SNPs in plasma cell myeloma.

Multi-drug resistance (MDR) leads to impaired treatment efficacy in all forms of malignancy. The main forms of MDR are thought to be mediated by the substrate transporting actions of certain adenosine triphosphate binding cassette (ABC) transport proteins. The genes ABCB1, ABCB4, ABCC1, ABCG2 and LRP1 have been identified as the most prominent contributors to clinically significant MDR. To date, no study has investigated the expression of these genes in plasma cell myeloma (PCM), or attempted to relate their expression to the incidence of relapse and/or stage at presentation. Here, we show that ABCB4 may be a prominent mediator of tumour cell MDR within PCM. Additionally, there are three SNPs (rs1045642, rs2032582 and rs1128503) within the most widely studied of these genes, ABCB1, which have been suggested to have a potential impact on OS in PCM and which may form a haplotype in ABCB1. rs1045642 in ABCB1 appears to be the only SNP affecting OS within the PCM patients studied, with minimal linkage disequilibrium demonstrated between it and rs2032582 and rs1128503.

Multidrug resistance in the chronic lymphoproliferative disorders.

Multidrug resistance (MDR) is a phenomenon in malignancy whereby tumor cells generate mechanisms to resist cytotoxic treatments. Several such mechanisms have been identified. However, to date the most significant research on MDR has involved the adenosine triphosphate binding cassette (ABC) transporter proteins, including P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP). These proteins have natural functions involving substrate transport in normal cells but are detrimental to treatment when expressed in the membrane of tumor cells. It remains unclear whether ABC mediated MDR functions primarily through protein up-regulation or via a relevant signaling mechanism, or is simply due to selective pressure on an already resistant tumor cell subpopulation. Here we present an overview of MDR in the chronic lymphoproliferative disorders (CLPDs), in particular that attributed to the ABC transporter protein family.

Vitamin D deficiency and insufficiency in pregnant women: a longitudinal study.

Maternal vitamin D insufficiency is associated with childhood rickets and longer-term problems including schizophrenia and type 1 diabetes. Whilst maternal vitamin D insufficiency is common in mothers with highly pigmented skin, little is known about vitamin D status of Caucasian pregnant women. The aim was to investigate vitamin D status in healthy Caucasian pregnant women and a group of age-matched non-pregnant controls living at 54-55 degrees N. In a longitudinal study, plasma 25-hydroxyvitamin D (25(OH)D) was assessed in ninety-nine pregnant women at 12, 20 and 35 weeks of gestation, and in thirty-eight non-pregnant women sampled concurrently. Plasma 25(OH)D concentrations were lower in pregnant women compared to non-pregnant women (P < 0.0001). Of the pregnant women, 35, 44 and 16 % were classified as vitamin D deficient (25(OH)D < 25 nmol/l), and 96, 96 and 75 % were classified as vitamin D insufficient (25(OH)D < 50 nmol/l) at 12, 20 and 35 weeks gestation, respectively. Vitamin D status was higher in pregnant women who reported taking multivitamin supplements at 12 (P < 0.0001), 20 (P = 0.001) and 35 (P = 0.001) weeks gestation than in non-supplement users. Vitamin D insufficiency is evident in pregnant women living at 54-55 degrees N. Women reporting use of vitamin D-containing supplements had higher vitamin D status, however, vitamin D insufficiency was still evident even in the face of supplement use. Given the potential consequences of hypovitaminosis D on health outcomes, vitamin D supplementation, perhaps at higher doses than currently available, is needed to improve maternal vitamin D nutriture.

ABCB1 (MDR1) rs1045642 is associated with increased overall survival in plasma cell myeloma.

Multi-drug resistance (MDR) may compromise the successful management of haematological malignancies, impairing the effectiveness of chemotherapy. The P-glycoprotein (P-gp) drug efflux pump, encoded by the gene ABCB1 (MDR1), is the most widely studied component in MDR. A single nucleotide polymorphism (SNP) has been identified within ABCB1, rs1045642 (C3435T), which may alter P-gp substrate specificity and have an impact on the effectiveness of treatment, and hence overall survival (OS). We estimated the frequency of this SNP in the Northern Irish population and investigated its impact on the OS of patients with plasma cell myeloma (PCM). There was no significant difference in the frequency of rs1045642 between the PCM cohort and an age- and gender-matched control population. Findings within the PCM cohort suggest that rs1045642 genotype influences OS (p = 2 x 10(-2)). If confirmed in larger studies, these results suggest that genotyping rs1045642 may be a useful predictor of outcome in PCM and could indicate modified treatment modalities in certain individuals.

Effect of zinc supplementation on the immune status of healthy older individuals aged 55-70 years: the ZENITH Study.

Aging is associated with alterations in the immune system, effects which may be exacerbated by inadequate zinc (Zn) status. We examined the relationship between Zn status and markers of immunity and the effect of supplementation with 15 mg or 30 mg Zn/d for 6 months on immune status in healthy individuals. Zn status was assessed by dietary intake and biochemical indices. Immune status was assessed by multiple flow cytometric methods. At baseline, Zn concentration was positively associated with lymphocyte subpopulation counts and T-lymphocyte activation. Zn supplementation of 30 mg/d significantly lowered B-lymphocyte count, albeit at month 3 only. Lower doses of Zn (15 mg Zn/d) significantly increased the ratio of CD4 to CD8 T lymphocytes at month 6. Overall, these findings suggest that total Zn intake (diet plus supplementation) of up to 40 mg Zn/d do not have significant long-term effects on immune status in apparently healthy persons aged 55-70 years.