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Genetic testing represents a major asset for discriminating between normal antigenic stimulation of B or T cells and malignant, pathological findings. The main difference between those two situations is the heterogenicity of lymphocyte populations as a result of diverse antigenic stimulation for the first situation, compared to single and identical population (clones) for the second situation. The present paper describes one of the many genetic assays capable of discriminating between normal and malignant, characterized by low time-to-result interval, accuracy, simplicity, and low cost comparing to other tests. [ABSTRACT FROM AUTHOR]

Transmembrane protease serine 4 (TMPRSS4) is a cancer-associated protease associated with prognosis in various types of cancer. Mechanistically, TMPRSS4 mainly regulates malignant phenotypes, such as tumor invasion and metastasis, by either the epithelial to mesenchymal transition (EMT) program or promoting the proliferation of cancer cells. To date, TMPRSS4 and immune infiltration in thyroid carcinoma (TC) are largely unknown. Thus, this paper evaluated the expression of TMPRSS4 in tumor tissue through the Tumor Immune Estimation Resource (TIMER) database, and Oncomine, and its correlation with clinical parameters by UALCAN databases. Furthermore, we analyzed its prognostic value from Kaplan-Meier Plotter database, and the relationship between TMPRSS4 and the abundance of tumor-infiltrating lymphocytes (TILs) in TC in TISIDB, screening potential immune targets to explore novel mechanisms for the clinical management of TC. Finally, we assessed the correlation between TMPRSS4 and some immune markers to uncover a potential immune-related biomarker in TC patients by TIMER2.0. The results revealed that TMPRSS4 was highly expressed in TC and was also associated with lymphatic metastasis, advanced stage, histological subtype, and favorable clinical outcome. The stratified analysis based on immune cell content showed that decreased TMPRSS4 had worse prognosis in CD8+ T cell-enriched TC patients. TMPRSS4 was positively correlated with tumor immune infiltration and the expression of gene markers of immune cells. Notably, its expression was lower in the lymphocyte-depleted subtype than in other immunosubtypes in TC. Moreover, TMPRSS4 was closely related to chemokines as well as their receptors and the immunosuppressive checkpoints CTLA-4, PD-1, and HLA-G. In conclusion, TMPRSS4 may act as a novel biomarker predicting prognosis and immune infiltration in TC. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) therapies are revolutionary cancer treatments; however, they only benefit about a third of patients. Therefore, extensive research is underway to find methods to improve their therapeutic efficacy. One avenue of study that has recently emerged is to consider the role the gut microbiome plays in therapeutic success. Several high‐impact studies have repeatedly shown that the presence, composition and level of diversity of the gut flora directly impact cancer treatment outcome in both mice and patients. These studies have also highlighted the danger of using antibiotics shortly before or during cancer treatments. However, there are still several questions that need to be answered, including which bacteria promote the greatest benefit, the mechanisms by which they act and how we can use this information to influence treatment outcome. In this review, we explain how the gut microbiome was realized to be of such importance and propose hypotheses for why gut flora have such a critical impact on ICB therapeutic success. We also describe a hypothetical mechanism involving bacterial translocation out of the gut and into the tumor, whereby the bacteria act in an adjuvant capacity to facilitate an antitumor response. By highlighting key papers in the field, we hope to hasten research on the subject so as to find a means to improve the therapeutic efficacy of these ground‐breaking cancer treatments. [ABSTRACT FROM AUTHOR]

Currently, immunomodulation is an alternative in the fight against many diseases, being considered a possibility to fight against many infectious diseases that can affect pig herds. Selenium can be used for this purpose, the effect of its administration on the immune response being the main purpose of the present study. Following the administration of organic selenium, we found that in the case of WBC parameters, granulocytes and agranulocytes percentages and lymphoblastic transformation percentages of B lymphocytes, there are no major differences between the values recorded at the beginning of the experiment and the values recorded in the two experimental moments. Instead, following the administration of organic selenium, we observed significant increases in T lymphocytes percentages (by 9.94%, after 21 days, and respectively by 8.18%, after 30 days), percentages of lymphoblastic transformation of T lymphocytes (by 59.59% after 21 days, and respectively by 64.14% after 30 days), as well as the helper T lymphocytes/supressor T lymphocytes ratio (by 46.15% after 21 days, and respectively by 65.38% after 30 days). Regarding the percentage of B lymphocytes, a decrease of this parameter is observed by 38.34% after 21 days and by 21.84% after 30 days following the administration of the product based on organic selenium. [ABSTRACT FROM AUTHOR]

It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1–10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms. [ABSTRACT FROM AUTHOR]

Aim and Objective: HIV (Human Immunodeficiency Virus) has been reported to weaken the immune system by binding to CD4+ receptor of T lymphocytes, thereby making the affected individual to become more susceptible to several microbial infections leading to life threatening problems. Recent literature indicate there is promsing advancement in identifying drug targets through CD4+ receptor binding capabilities with its ligand. The main purpose of this paper was to study the various molecular interactions of HIV to other molecules like receptors CD4+ and also co-receptors like CCR5 and CXCR4. Materials and Required: The molecules are going to be modeled by using software known as Swiss PDB viewer. The first objective is to get the FASTA sequence from the NCBI website. Then we need to paste the sequence onto the Swiss PDB viewer. Conclusion: The requierd protein/molecule is modeled. We are going to follow this method to obtain templates and modles for the following molecukles: HIV, GP120, CCR5 and CXCR4. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are characterized by a semi-invariant TCR that recognizes vitamin B metabolite Ags presented by the MHC-related molecule MR1. Their Ag restriction determines a unique developmental lineage, imbuing a tissue-homing, preprimed phenotype with antimicrobial function. A growing body of literature indicates that MR1-restricted T cells are more diverse than the MAIT term implies. Namely, it is increasingly clear that TCR a- and TCR b-chain diversity within the MR1-restricted repertoire provides a potential mechanism of Ag discrimination, and context-dependent functional variation suggests a role for MR1-restricted T cells in diverse physiological settings. In this paper, we summarize MR1-restricted T cell biology, with an emphasis on TCR diversity, Ag discrimination, and functional heterogeneity. [ABSTRACT FROM AUTHOR]

Background: Paracoccidioidomycosis, a chronic granulomatous fungal disease caused by Paracoccidioides brasiliensis yeast cells affects mainly rural workers, albeit recently cases in immunosuppressed individuals has been reported. Protective immune response against P. brasiliensis is dependent on the activity of helper T cells especially IFN-γ-producing Th1 cells. It has been proposed that Paracoccidioides brasiliensis is able to modulate the immune response towards a permissive state and that the thymus plays a major role in it.Methods: In this paper, we show that acute infection of BALB/c mice with P. brasiliensis virulent isolate (Pb18) might cause alterations in the thymic environment as well as the prohibitive TCR-expressing T cells in the spleens.Results: After seven days of infection, we found yeast cells on the thymic stroma, the thymic epithelial cells (TEC) were altered regarding their spatial-orientation and inflammatory mediators gene expression was increased. Likewise, thymocytes (differentiating T cells) presented higher migratory ability in ex vivo experiments. Notwithstanding, P. brasiliensis-infected mice showed an increased frequency of prohibitive TCR-expressing T cells in the spleens, suggesting that the selection processes that occur in the thymus may be compromised during the acute infection.Conclusion: In this paper, for the first time, we show that acute infection with Paracoccidioides brasiliensis yeast cells promotes thymic alterations leading to a defective repertoire of peripheral T cells. The data presented here may represent new mechanisms by which P. brasiliensis subverts the immune response towards the chronic infection observed in humans. [ABSTRACT FROM AUTHOR]

Immunomodulation is an important alternative in combating many diseases, being considered a potential weapon in the fight against pathological entities that cause major economic losses in pig herds. Levamisole, in addition to its anthelmintic effect, has also an immunostimulatory effect, for which it has also been used as a vaccine adjuvant. The aim of the research was to evaluate the non-specific immune response in modern techniques in pigs given levamisole. The results showed significant differences in the case of the ratio of lymphocyte subpopulations, there was an increasing trend in favor of T lymphocytes (with 10.44%), and the % of T lymphocytes blastic transformation (with 56%). Also, we observed a significant increase of the LTh/LTs ratio (with 65.92%) which supports the immunomodulatory potential of the levamisole and its involvement on the coordination of immune processes. [ABSTRACT FROM AUTHOR]

Cross-presentation of apoptotic cell associated antigens by immature dendritic cells prevents the activation of self reactive CD8 T cells. Tolerized self reactive CD8 T cells down-regulate IL-7R expression on their surface. Whether over-expression of IL-7R can reverse their fate and function has not been examined. In this paper, we showed forced expression of IL-7R in OT-I T cells by a transgene enhanced CD8 T cell mediated diabetes in the RIP-mOVA model. Although IL-7R Tg (transgenic) did not completely reverse the deletion of OT-I T cells, it provided a significant survival advantage over w.t OT-I T cells. Furthermore, IL7R Tg OT-I T cells isolated from diabetic pancreata displayed increased production of IFN-γ, higher expression of T-bet, and increased externalization of CD107a. We also found that immature DCs containing apoptotic cells expressed high levels of PDL-1 on their surface. Although IL-7R Tg did not change PD1 expression on activated OT-I cells in vivo, the transgene enabled a significantly lower number of OT-I T cells to induce diabetes in the absence of PDL-1. Our results demonstrated that forced expression of IL-7R not only improved the functionality of tolerized CD8 T cells, it also acted in synergy with PDL-1 deficiency to further promote CD8 T cell cytotoxicity to self antigens. [ABSTRACT FROM AUTHOR]

Estimating the time since infection (TI) in newly diagnosed HIV-1 patients is challenging, but important to understand the epidemiology of the infection. Here we explore the utility of virus diversity estimated by next-generation sequencing (NGS) as novel biomarker by using a recent genome-wide longitudinal dataset obtained from 11 untreated HIV-1-infected patients with known dates of infection. The results were validated on a second dataset from 31 patients. Virus diversity increased linearly with time, particularly at 3rd codon positions, with little inter-patient variation. The precision of the TI estimate improved with increasing sequencing depth, showing that diversity in NGS data yields superior estimates to the number of ambiguous sites in Sanger sequences, which is one of the alternative biomarkers. The full advantage of deep NGS was utilized with continuous diversity measures such as average pairwise distance or site entropy, rather than the fraction of polymorphic sites. The precision depended on the genomic region and codon position and was highest when 3rd codon positions in the entire pol gene were used. For these data, TI estimates had a mean absolute error of around 1 year. The error increased only slightly from around 0.6 years at a TI of 6 months to around 1.1 years at 6 years. Our results show that virus diversity determined by NGS can be used to estimate time since HIV-1 infection many years after the infection, in contrast to most alternative biomarkers. We provide the regression coefficients as well as web tool for TI estimation. unfortunately, they only distinguish between recent and long-term infections (concentration of HIV-1-specific antibodies) or are imprecise (immune status as measured by levels of CD4+ T-lymphocytes and viral sequence diversity measured by polymorphisms in Sanger sequences). In this paper, we show that recent advances in sequencing technologies, i.e. the development of next generation sequencing, enable significantly more precise determination of the time since HIV-1 infection, even many years after the infection event. This is a significant advance which could translate into more effective HIV-1 prevention [ABSTRACT FROM AUTHOR]

Endometriosis is a benign, chronic inflammatory disease that presents alterations in immune response that can be detected in eutopic endometrium, peritoneal fluid and peripheral blood of affected women. Regulatory T (T Reg ) cells are a subpopulation of T lymphocytes specialized in immune regulation that seem to participate in the development of endometriosis, by suppressing the immune response and favoring the establishment of lesions. Our aim was to review the scientific literature that evaluates T Reg cell phenotypes in the context of endometriosis. PRISMA statement for systematic reviews was applied, using “regulatory T cells” and “endometriosis” as keywords in the following databases: PubMed, Cochrane, EMBASE and Lilacs. The initial search and abstract review yielded 41 papers relating to the subject. At the end, 12 studies, published between 2009 and 2016, were included. Most studies that analyzed T Reg cells did not characterize these cells with current Bona Fide markers. In peritoneal fluid and endometriotic lesions, there was a higher concentration of T Reg cell phenotype and/or T Reg cell expression markers in patients with endometriosis when compared with controls. However, there is still not a consensus about T Reg cells concentration in eutopic endometrium and peripheral blood between the revised studies. Taken together, this data collection suggests that endometriosis is related to T Reg cells alterations, although further studies are necessary to reach more precise conclusions, especially regarding the percentage of these cells in eutopic endometrium and peripheral blood. This systematic review attempted to provide instructive and up-to-date collection of data that may help better design future studies. [ABSTRACT FROM AUTHOR]

Background: The B and T cells of the human adaptive immune system leverage a highly diverse repertoire of antigen-specific receptors to protect the human body from pathogens. The sequencing and analysis of immune repertoires is emerging as an important tool to understand immune responses, whether beneficial or harmful (in the case of autoimmunity). However, methods for studying these repertoires, and for directly comparing different immune repertoires, are lacking. Results: In this paper, we present a non-parametric method for directly comparing sequencing repertoires, with the goal of rigorously quantifying differences in V, D, and J gene segment utilization. This method, referred to as the Repertoire Dissimilarity Index (RDI), uses a bootstrapped subsampling approach to account for variance in sequencing depth, and, coupled with a data simulation approach, allows for direct quantification of the average variation between repertoires. We use the RDI method to recapitulate known differences in the formation of the CD4+ and CD8+ T cell repertoires, and further show that antigen-driven activation of naïve CD8+ T cells is more selective than in the CD4+ repertoire, resulting in a more specialized CD8+ memory repertoire. Conclusions: We prove that the RDI method is an accurate and versatile method for comparisons of immune repertoires. The RDI method has been implemented as an R package, and is available for download through Bitbucket. [ABSTRACT FROM AUTHOR]

The koala ( Phascolarctos cinereus ) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4 + lymphocytes collected from koala spleens (41.1%, range 20–45.1%) lymph nodes (36.3%, range 19–55.9%) and peripheral blood (23.8%, range 17.3–35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4 + cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4 + T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4 + T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system. [ABSTRACT FROM AUTHOR]

The aim of this paper was to look for predictors of abatacept (ABA) therapy discontinuation in patients with rheumatoid arthritis (RA). Seventy-one RA patients treated with ABA were followed up. Demographical, clinical, and laboratory parameters of the patients, including peripheral blood T and B cell populations, different rheumatoid factor and anti-cyclic citrullinated peptide autoantibodies isotypes, and serum free light chains were evaluated. Comparing patients who discontinued ABA with those still in therapy we observed: a higher proportion of smokers (51.9 vs 25.6 %; p = 0.03); a non significant lower proportion of anti-cyclic citrullinated peptide positivity (76 vs 89.5 %; p = 0.13); a lower proportion of terminally differentiated effector memory cells (TDEM) among total CD8+ T lymphocytes at baseline (22.0 % (7.8-39.2) vs 38.7 % (20.7-55.9); p = 0.002). Logistic multivariate analysis showed that only the proportion of CD8+TDEM T cells was an independent predictive factor of therapy discontinuation (OR (95 % IC) = 6.2 (1.2 to 30.8); p = 0.026). Receiver-operating characteristic analysis showed a significant performance of this biomarker for prediction of therapy discontinuation (using a cut-off of 30.6 %: AUC: 0.760 ± 0.07; p = 0.002). Patients with a low proportion of CD8+TDEM at baseline had a higher probability of discontinuing the treatment during time (log-rank test: p < 0.01). T cell characterization for identification of TDEM CD8+ T cells might be a useful test to predict discontinuation of ABA therapy. [ABSTRACT FROM AUTHOR]

Background: Intratumoural lymphocytic infiltration is strongly associated with the outcome of many human epithelial cancers. The current paper investigated whether subpopulations of tumour-infiltrating T lymphocytes are associated with certain clinicopathological parameters and the prognosis of patients with invasive bladder cancer (BCa). Patients and methods: The infiltration densities of the adaptive immune markers CD3 (the whole T cell population), FOXP3 (regulatory T cells; Tregs), CD8 (T effector cells) and CD45R0 (T effector memory cells) were analysed by immunohistochemistry and image analysis with tissue microarrays of tumour tissues from 149 patients with invasive BCa treated with radical cystectomy. The findings were correlated with certain clinicopathological parameters. Results: Higher FOXP3/CD3 [OS: p = 0.016, HR 1.29, 95 % confidence intervals (95 % CIs 1.05-1.59)] and FOXP3/CD8 (OS: p = 0.013, HR 1.32, 95 % CIs 1.06-1.65) ratios were significantly associated with briefer overall survival and time to cancer-specific death; the latter ratio represented an independent prognostic factor according to a multivariate analysis adjusted for pathological T and N stages (HR 1.32, 95 % CIs 1.05-1.67, p = 0.018). The infiltration densities of individual markers (CD3, CD8, FOXP3 and CD45R0) were not significantly associated with clinicopathological parameters or survival; however, a trend towards a better outcome was observed for higher log-transformed CD8 ( p = 0.070, HR 0.80, 95 % CIs 0.63-1.02) and CD3 ( p = 0.113, HR 0.84, 95 % CIs 0.68-1.04) infiltration values. Conclusions: A high fraction of Tregs amongst CD3- and CD8-positive lymphocytes indicated a poor prognosis, thereby emphasising the important role that Tregs play in the suppression of the anti-tumour immune response. No single lymphocytic marker was significantly correlated with clinical outcomes, but high CD3 and CD8 infiltration showed trends towards better prognosis. [ABSTRACT FROM AUTHOR]

Telomeres are essential in maintaining chromosome integrity and in controlling cellular replication. Attrition of telomere length in peripheral blood mononuclear cells (PBMCs) with age is well documented from cross-sectional studies. But the actual in vivo changes in telomere lengths and its relationship with the contributing factors within the individuals with age have not been fully addressed. In the present paper, we report a longitudinal analysis of telomere length in the PBMCs, lymphocytes and monocytes of 216 human subjects aged from 20-90 years assessed at 0-, 5- and 12-year follow-up. For the 5- and 12-year follow-up, telomere length in the PBMCs decreased in 34% and 46%, exhibited no detectable change in 56% and 47% and increased in 10% and 7% of the subjects respectively. The rate of telomere change was distinct for T-cells, B-cells and monocytes for any given subject. Telomerase activity declined with age in the resting T-cells and B-cells and the activated T-cells. Finally, a significant portion of telomere attrition in T-cells with age was explained by a decline in the telomerase activity, decreased naıve cells and the change in physiological conditions such as elevated blood glucose and interleukin (IL)-6 levels. These findings show that changes in the telomere length of the PBMCs with age in vivo occur at different rates in different individuals and cell types and reveal that changes in the telomere length in the T-cells with age is influenced by the telomerase activity, naïve T-cell percentage and changes in health conditions. [ABSTRACT FROM AUTHOR]

Lymphocyte invasion by HIV-1 is a complex, highly regulated process involving many different types of molecules that is prompted by the virus's association with viral receptors located at the cell-surface membrane that culminates in the formation of a fusion pore through which the virus enters the cell. A great deal of work has been done to identify the key actors in the process and determine the regulatory interactions; however, there have been no reports to date of attempts being made to fully understand the system dynamics through a systemic, quantitative modeling approach. In this paper, we introduce a dynamic mathematical model that integrates the available information on the molecular events involved in lymphocyte invasion. Our model shows that moesin activation is induced by virus signaling, while filamin-A is mobilized by the receptor capping. Actin disaggregation from the cap is facilitated by cofilin. Cofilin is inactivated by HIV-1 signaling in activated lymphocytes, while in resting lymphocytes another signal is required to activate cofilin in the later stages in order to accelerate the decay of the aggregated actin as a restriction factor for the viral entry. Furthermore, stopping the activation signaling of moesin is sufficient to liberate the actin filaments from the cap. The model also shows the positive effect of gelsolin on actin capping by means of the nucleation effect. These findings allow us to propose novel approaches in the search for new therapeutic strategies. In particular, gelsolin inhibition is seen as a promising target for preventing HIV-1 entry into lymphocytes, due to its role in facilitating the capping needed for the invasion. Also it is shown that HIV-1 should overcome the cortical actin barrier during early infection and predicts the different susceptibility of CD4+ T cells to be infected in terms of actin cytoskeleton dynamics driven by associated cellular factors. [ABSTRACT FROM AUTHOR]

Although regulatory T cells protect people from autoimmunity, two recent papers in Immunity ( Malchow et al., 2016; Kieback et al., 2016 ) demonstrate that these cells are also a crisis averted. Without the proper education in the thymus, these cells will turn on their host and cause autoimmunity. [ABSTRACT FROM AUTHOR]

Natural Killer (NK) cells are innate lymphocytes that contribute to immune protection by cytosis, cytokine secretion, and regulation of adaptive responses of T cells. NK cells distinguish between healthy and ill cells, and generate a cytotoxic response, being cumulatively regulated by environmental signals delivered through their diverse receptors. Recent advances in biomaterials and device engineering paved the way to numerous artificial microenvironments for cells, which produce synthetic signals identical or similar to those provided by the physiological environment. In this paper, we review recent advances in materials and devices for artificial signaling, which have been applied to regulate NK cells, and systematically study the role of these signals in NK cell function. [ABSTRACT FROM AUTHOR]

The article reports on findings of two research papers including one on T lymphocyte with restricted antigen receptor diversity by Masaru Taniguchi & others, and another on human cells by Steven Balk & colleagues. Topics include the effects of invariant natural killer T cells on immune system, use of denaturing gel electrophoresis, polymerase chain reaction, and cloning for sequencing two invariant T-cell receptor (TCR) rearrangements & discovery of lymphocytes with restricted TCR diversity.

The article discusses a research paper on the use of normalization of CD4 T cell metabolism for the treatment of lupus. It references the study "Normalization of CD4 T Cell Metabolism Reverses Lupus," published by Y. Yin et al., published in an issue of "Science Translational Medicine." It discusses the dependence of T-cell function on cellular metabolism, the response of polyclonal stimulation in vitro and findings on the extracellular acidification rate and oxygen consumption rate.