In other words, the immune system must allow generation of autoreactivity to occur to eliminate the cancer cells. Results of studies in cancer immunology are challenging the old concept that the immune system is tightly regulated, not allowing for reactivity to self. Instead, new concepts illustrate that the immune system is not so tightly regulated to prevent reactivity to self; rather, the normal immune repertoire

consists of both T cells and B cells capable of recognizing self [5–9]. However, under most normal circumstances the immune system’s regulatory mechanisms are effective in maintaining control over the autoreactive cells preventing the development of autoimmune disease while maintaining the immunosurveillance necessary to avoid establishment of malignancies. selleckchem A delicate balance exists in the multi-faceted normal immune system encompassing effector mechanisms designed to initiate inflammatory this website and autoreactivity balanced against regulatory mechanisms

designed to control both inflammatory and autoimmune responses and protect the host from subsequent damage. Some of the challenges for medicine are to induce potent tumour immunity (autoreactivity) balanced against the risk of development of autoimmune disease and to establish effective inflammatory responses to rid the host of assaulting pathogens without allowing for chronic inflammatory conditions which may lead to subsequent inflammatory disease. Another emerging area of intriguing data points to the ageing immune system as a potential cause of chronic inflammatory and/or autoimmune disease development. As the host ages the immune system, like many organ systems, experiences either diminished or loss of functional capacity. This concept of autoimmunity proposes that the failure of control mechanisms as the host ages may be a primary risk factor for autoimmune disease development in older individuals [9].

Inflammatory and autoimmune responses are therefore part of the normal and protective capabilities of the host’s immune system. However, when Dapagliflozin does the inflammation become chronic, escalating from an inflammatory condition to an inflammatory disease, or when does the autoreactivity become autoimmune disease? In the remainder of this review, we will focus on the concepts of inflammatory and autoimmune responses in association with the development of type 2 diabetes. Diabetes mellitus is a spectrum of diseases encompassing type 1 (T1D) and type 2 (T2D) diabetes [10–12]. The diagnosis of T1D versus T2D is commonly made using criteria such as age at onset, abruptness of hyperglycaemic symptoms, presence of ketosis, degree of obesity and the perceived need for insulin replacement.

[109] Pre-eclampsia is a pregnancy-related syndrome that see more affects multiple systems and clinically presents as hypertension, proteinuria, edema, and in its more sever forms evidence

of fetal compromise, neurologic abnormality, liver and hematologic dysfunction.[110] The complexity of the syndrome defies the development of a panel of genetic screens or biomarkers.[111] While the basic cause of the disease is as yet unknown, multiple hypotheses exist. These include failure of placentation[112] and thus reduced utero-placental perfusion, intolerance to volume expansion generated by pregnancy,[113] infection,[114] and inflammation.[115] It is hotly debated as to whether failed placentation is caused or a by-product of broken maternal immune tolerance.[116, 117] Many agree that a common final pathway to the manifestation of the disease is endothelial cell damage occurring in a variety of vascular beds.[118] While the Regorafenib chemical structure disease is thought of as being unique in human, many recognize the potential positive role of the integration of research in human and animal models in understanding the underlying mechanisms.[119, 120] The hallmarks of pre-eclampsia most sought

after in animal models are hypertension, renal dysfunction (proteinuria), and further, conditions such as poor trophoblast invasion and endothelial damage. Current models address some of these issues. There have been rare reports of spontaneous pre-eclampsia in related non-human primates.[121] These species have also been used to develop models of pregnancy-related hypertension and proteinuria based on injection during mid-gestation of inflammatory mediators, such as tumor necrosis factor[122] or antibodies to interleukin 10.[123] There are strains of mice that spontaneously develop hypertension, proteinuria, smaller litters, and fetal demise, and these have been used to model pre-eclampsia.[124, 3-mercaptopyruvate sulfurtransferase 125] There are also models of spontaneous pregnancy-associated hypertension with fetal compromise

in rats.[126] There also exist genetically manipulated mouse and rat models. In one interesting genetic model of hypertension in pregnancy, female mice transgenic for human angiotensinogen are mated to males transgenic for human rennin.[127] The resulting pregnancy is marked by distortion of placental anatomy, elevation of circulation vascular endothelial growth factor (VEGF) receptor in mid-gestation (12–13 of 19–20 days), hypertension, fetal intrauterine growth retardation, and systemic maternal disorders including proteinuria and convulsion. In the rat version of this model,[128] the hypertensive disease experienced by the pregnant rat is thought related to secretion of rennin from the placenta into the maternal circulation.

Moreover, combination therapy using cisplatin and human leucocyte antigen-A24-restricted human vascular endothelial growth factor receptor 1 (VEGFR1)-1084 and VEGFR2-169 in patients with advanced or recurrent adenocarcinoma of the stomach showed that the disease control rate (partial and stable disease) was 100% after two cycles of the combination therapy [25]. Delayed-type hypersensitivity response to leishmanial antigens has been widely used to assess the level of host protection to the disease [26]. It has been well established that induction of a DTH response is mediated via Th1 cell as it secretes IFN-γ which

is expressed during macrophage stimulation Selumetinib purchase for parasite killing [27]. The DTH responses to leishmanin were apparent during L. donovani infection in BALB/c mice as evident by an increase in the foot pad swelling after injection of leishmanin. The increase was much higher when the animals were treated with immunochemotherapy than the groups KPT-330 chemical structure of animals treated with

chemotherapy or immunotherapy alone. This suggests that the mice treated with cisplatin + 78 kDa with or without adjuvant (MPL-A) developed a strong cell-mediated immune response indicating that drug treatment followed by vaccine therapy was helpful in reversal of immunosuppression caused by the parasite. Earlier studies from our laboratory reported an increased DTH response in animals treated with low dose of cisplatin [14]. Correlation between DTH responses and parasite load has also been reported [14, 15]. This was evident from our results where a strong positive correlation was observed between enhanced DTH response and reduced parasite load. The immunological response was further characterized by analysing the

distribution of IgG1 and IgG2a specific antibodies in the serum samples of infected and treated BALB/c mice. Production of IgG2a is normally associated with IFN-γ secretion and the development of a Th1 immune response. However, in contrast, production of the IgG1 is normally associated with IL-4 secretion and the development of Th2 type of response. The treated animals revealed higher IgG2a and lower IgG1 levels than the infected controls. However, maximum levels of IgG2a and minimum levels of IgG1 were observed in animals DNA ligase treated with cisplatin + 78 kDa + MPL-A than those animals that are treated with cisplatin alone or 78 kDa/78 kDa + MPL-A alone. It has been shown earlier from our laboratory that immunization of mice with 78 kDa + MPL-A resulted in significant increase in IgG2a response [6]. Moreover, a significant reduction in specific antibody titres was observed after treatment with immunochemotherapy (Glucantime + Leish-110f/MPL-SE) in dogs suffering from canine leishmaniasis [18]. Th1 and Th2 cell lymphocytes are important mediators in generating immunity to leishmaniasis and can be distinguished by the cytokines they secrete.

Then we tested for acquired immunity by comparing worm burdens in the immunized-challenged hamsters (Group 5) and the challenge controls (Group 4), with a specific prediction that Group 4 would have more worms than Group 5. The Mann–Whitney

U test was used post hoc in SPSS to explore differences in worm burden between specified groups. All other quantified parameters of the mucosal response to infection were examined by selleck kinase inhibitor general linear models (GLM) in SPSS (version 12.0.1 for Windows) fitting treatment (the five treatments) and time (days 73 and 94 of the experiment, excluding the values derived from Group 5 hamsters culled on days 80 and 87). Models were scrutinized carefully for approximately normal distribution of residuals. In Group 5 hamsters (primary + secondary infection), for which data were derived on four separate days (73, 80, 87 and 94 of the experiment), we additionally looked for changes over time. If the data appeared approximately linearly distributed, we employed parametric regression analysis (Pearson’s) in SPSS, with days of the experiment as the independent factor. For nonlinear trends, we fitted the best-fit curves in SPSS, and tested them for goodness of fit by F tests. The mean worm burden of each experimental

group at autopsy is shown in Table 2. Not surprisingly the naïve control group (Group 1), and the group treated with ivermectin on day 35 post-infection X-396 supplier (p.i.) (Group 3, primary abbreviated infection) were without worms at autopsy. Group 2 (primary continuous infection), had low worm burdens on days 73 and 94 p.i., with some adult worms still persisting from the original immunizing infection given on day 0, but representing a stable infection: there was no statistically significant difference between mean worm burdens in Group 2 hamsters Tau-protein kinase on day 73 and 94 (Mann–Whitney U test, z = 0·7). The challenge control group (Group 4), given only the second

infection, had higher worm burdens than the immunized-challenged group (Group 5, primary + secondary infection; 2-way anova, confined to Groups 4 and 5, and days 10 and 31 post-challenge infection (p.c.), for the specific prediction, z = 2·72, P = 0·0033), indicating that Group 5 had expressed acquired resistance to challenge. The results are illustrated in Figure 1, and the statistical analysis is given in the legend. Naïve control hamsters (Group 1) maintained the height of villi between the two sampling points (Figure 1; days 73 and 94 from the start of the experiment) and the values recorded were within, albeit towards the lower end of, the normal range reported earlier from naive hamsters (20). Hamsters infected on day 0 of the experiment and sustaining a continuous infection throughout (Group 2, primary continuous infection), had villi with drastically reduced height on both days, with values not atypical of those reported by Alkazmi et al. (20).

This study would be the first of its kind in the field

of human VL, as none of the reports dealt with human iNKT cells and their dynamics with the therapy. A belief was that CD1d-reactive cells must be expressing invariant TCR (Vα24 and Vβ11 in human) (1–3). In contrast, our finding suggests that all invariant TCR-expressing cells (iNKT) may not be solely reactive to the CD1d, especially in diseased condition, as evident from their proportional frequency (15). If CD1d-reactive NKT cells are approximately 0·2–0·4% of total lymphocyte, then what could be the reactivity of remaining iNKT cells? (approximately 1% of total lymphocyte). It indicates that all iNKT cells may not be CD1d LEE011 reactive. Definition of human iNKT cells is further compounded by the strange observation

of a novel population with Vβ11+, but CD161− (Figure 1b,d). This population may be expressing other NK cell marker of NK cell recognition complex. learn more But absence of a stimulatory/activating receptor CD161, which recognizes non-MHC ligand, potentiates possible function of this novel subset in a MHC-dependent manner. However, a detailed study is required in this regard. Duality in function of the enriched iNKT cells at the disease site will be crucial in dictating the disease outcome. Dichotomies in the functional behaviour of iNKT cells are in support for the existence of iNKT-1 (IFN-γ producing) and iNKT-2 (IL-4 producing), very similar to Th-1 and Th-2 (16). The antigen-specific response of these functionally divergent cells will be relevant in context of the pathology of VL. They may have some role in the early modulation/triggering of forthcoming immune response at disease site. This study was supported by Department of Biotechnology (Government Masitinib (AB1010) of India) for funding the work (Grant No.: BT/PR6737/Med/14/871/2005).

In addition, we thank the Council of Scientific and Industrial Research (CSIR), Government of India, for providing fellowship to Dr. Ambak K. Rai. The authors thank all the patients and control subjects who voluntarily agreed to participate in this study. We also thank Dr. Pradeep Dagur and Dr. Beenu Joshi, (JALMA, Agra, India) for helping in preparation of Leishmania antigen and extremely grateful to Dr. R. Viswakarma (NII, New Delhi, India) for providing LPG. Figure S1. MNCs derived from blood of patients were stained under various staining conditions (a) Preloaded CD1d dimer with aGalcer, but no secondary fluorescent antibody, (b) unloaded CD1d dimer, with secondary fluorescent antibody and (c) preloaded CD1d dimer with aGalcer, with secondary fluorescent antibody. Figure S2. MNCs were incubated with CD1d dimer under following conditions (a) Loaded CD1d dimer, no aIgG1 FITC, (b) unloaded CD1d dimer, aIgG1 FITC, (c) Loaded CD1d dimer (in 10× molar access), aIgG1 FITC and (d) Loaded CD1d dimer (in 20× molar access), aIgG1 FITC. Figure S3.

Serum NGF levels varied greatly. Serum

NGF concentrations in healthy humans are not normally distributed. About 10% of healthy people have relatively high NGF concentrations.68,69 We also noted the same findings of serum NGF levels in OAB patients, but not in normal controls. The high serum NGF levels in healthy humans in other studies might result from underlying systemic conditions that affect serum NGF levels. C-reactive protein (CRP) is a protein found in the blood, the levels of which rise in response to inflammation. CRP is synthesized by the liver in response to factors released by fat cells (adipocytes).70 Serum CRP level can be used as a nonspecific marker of systemic inflammation. Chronic prostatic inflammation has been hypothesized to be associated with the NVP-BGJ398 datasheet pathogenesis of benign prostatic hyperplasia. However, the association between histological prostatic inflammation and LUTS is relatively weak.71 Rohrmann et al.72 reported that men with serum CRP levels >0.30 mg/dL were more likely to show three or four symptoms

(i.e. nocturia, incomplete emptying, hesitancy, and weak stream) from the Third National RG7420 in vivo Health and Nutrition Examination Survey (NHANES III). Another report using longitudinal data from the Olmsted County study73 showed that patients with higher serum CRP levels were approximately two times more likely to exhibit a rapid increase in storage LUTS and almost 2.5 times more likely to show a rapid decrease in peak flow rate. Kupelian et al.74 reported a significant association between serum CRP level and overall International Prostate Symptom Score (IPSS) in both men and women included in the Boston Area Community

Health (BACH) survey. We Rolziracetam also reported the serum CRP levels are associated with residual urgency symptoms in patients with benign prostatic hyperplasia after medical treatment.75 In women, serum CRP was also found to elevate in OAB patients. CRP levels were significantly higher in women with OAB-wet than in those with bladder oversensitivity and in the normal control group. Women with voiding dysfunction also had a non-significantly higher CRP level. Further analysis revealed that body mass index and maximum flow rate were two independent factors influencing CRP levels. However, serum CRP level is not considered a suitable biomarker for discriminating female non-SUI LUTD. As patients with OAB may have frequent detrusor contractions during the storage phase, it is possible that sustained isometric detrusor contractions could result in increased muscle bulk and hence increased detrusor wall thickness (DWT) or bladder wall thickness (BWT). It has been hypothesized that DWT increases in patients with DO.

The TST was performed by trained personnel on all study participants, using PPD as the antigen, in accordance with the standard intradermal Mantoux method protocol. The test reading was conducted 72 h after the subcutaneous injection, based on the size of induration measured. The individuals were scored as non-reactive (0–4 mm), low reactive (5–9 mm) and strongly reactive (>10 mm). The study protocol was approved by the Ethics Committee of the Centro de Pesquisas Aggeu Magalhães – FIOCRUZ (number 55/02) and by the Instituto Materno Infantil Professor Fernando Figueira, and informed consent was obtained from the parents or legal representatives of the participants.

Cell preparation and culture.  Blood samples (3 ml) were taken with heparin (10 U/ml) by venipuncture. The whole blood was cultivated in an RPMI 1640 medium with penicillin/streptomycin (100 U/ml, 100 μg/ml) and incubated with ESAT-6 (3 μg/ml), CFP-10 (3 μg/ ATM inhibitor ml), PPD (5 μg/ml) or PMA/Iono (Phorbol Miristate Acetate, 5 μg/ml/ Ionomicin, 1 μg/ml) at 37 °C in a humidified CO2 atmosphere for 120 h. This time period was chosen after kinetic selleck inhibitor study of INF-γ. The supernatants were harvested and immediately frozen at −70 °C until analysis. ESAT-6

and CFP-10 were obtained by donations from FIOCRUZ and Statens Serum Institute (Copenhagen, Denmark), respectively. PPD in vitro (1 mg/ml) was commercially obtained by FIOCRUZ. The interferon-γ release assay.  The concentration of IFN-γ in duplicate samples was determined using the Quantikine kit (R&D Systems, Minneapolis, Cytidine deaminase MN, USA) ELISA (enzyme-linked immunosorbent assay) as described in the manufacturer’s instructions, and the results were processed using Microplate Manager, version 4.0 (BIORAD laboratories, Hercules, CA, USA) and expressed as pg/ml with detection limits ranging from 15.6 to 1000.00 pg/ml. Statistical analysis

and determination of sensitivity and specificity.  The differences between the mean IFN-γ levels of the groups were evaluated using an unpaired Student’s t-test. P values of <0.05 were considered significant. The receiver operating characteristic (ROC) curve, cut-off, sensitivities and specificities for each antigen were estimated using the specific spss Base software, version 13 (Chicago, IL, USA), with a confidence interval of 95%. The areas under the curve (AUC) show the sensitivity versus 1-specificity, having values between 0.5 and 1.0, with those closer to 1.0 possessing better discriminatory power. The Kappa statistic represents the level of agreement between the clinical classifications of the children and the test results and was obtained using Epi Info, Version 6.04 (Centers for Disease Control and Prevention, Atlanta, GA, USA). The likelihood ratios for each test were calculated as described by Sackett et al.

Our experimental approach might be useful for addressing these issues. Unfortunately, however, we were unable to characterize the CD4-reactive Ab-producing cells, as the oligoclonal cultures of B-LCL were terminated after RNA extraction for our Ig gene cloning strategy. We speculate that B-1 cells could be the source

of the CD4-reactive Ab, because B-1 cells produce IgM that often cross-reacts with auto-Ag. Our genetic data indicated that only a fraction of the CD4-reactive Ab could have some HIV-inhibitory function. It is an open question whether such CD4-reactive HIV-inhibitory Ab may be present in the other healthy individuals, as well as in HIV-seropositive long-term non-progressors. HIV-inhibitory CD4-reactive Ab are effective against multiple HIV clades, as CD4 is the major HIV receptor buy LDK378 for all the viral clades 11. A clinical trial is being conducted to examine the therapeutic efficacy of a humanized CD4-reactive mAb in patients with HIV infection 8, 12. Although CD4-reactive

Ab can be detected HIF-1�� pathway in healthy individuals, safety is always a concern when using self-recognizing Ab as therapeutic drugs. Given that HO538-213 was isolated from a healthy individual and that it recognized a different epitope than Leu-3a, HO538-213 might effectively inhibit HIV without disturbing CD4+ T-cell functions. As noted above, the donor from which the three CD4-reactive IgM Fab were isolated has been healthy for more than 29 years since PBMC collection, suggesting that these Ab may not seriously inhibit CD4+ T-cell functions in vivo and thus may be useful in treating HIV infection and other disorders 4. This report provides the first clonal genetic analyses of human monoclonal anti-CD4 Ab. IgM is considered

to function in “natural humoral immunity”, as it has a relatively low affinity for pathogens and confers natural resistance to infectious agents. However, the pathogen-specific immunity function of IgM has not been Megestrol Acetate demonstrated at a clonal level. Our data suggest that CD4-reactive IgM is present in healthy individuals and can contribute to natural resistance to HIV infection and AIDS progression. This is the first clear demonstration of a natural humoral immunity function of IgM against HIV. The establishment of Ab-producing cells, cloning of Ig genes encoding V regions, ELISA, and the purification of Fab fragments from Escherichia coli have been described previously 16. The experimental procedure is schematically shown in the Supporting Information Fig. 1. In brief, PBMC from 12 donors, including two healthy individuals and ten individuals with autoimmune disorders, were infected with the B95-8 strain of EBV, and 1×104 cells were propagated in 96-well plates. The supernatant was analyzed by ELISA using rhCD4 derived from a baculovirus system (50 ng/well; INTRACELL) as an Ag.

We replaced one copy of ERG11 with ERG11 containing the T916C mutation in C. albicans CAI4 and expressed ERG11 with the T916C mutation in Saccharomyces cerevisiae INVSc1. The MIC values were two- to four-fold greater in CAI4 transformants with than without the T916C mutation and 128 and 32 μg ml−1 for S. cerevisiae INVSc1-containing ERG11 with and without the T916C mutation. T916C mutation may Alvelestat mw be associated with fluconazole resistance in C. albicans. “
“The State of Ceará in north-eastern Brazil has one of the highest rates in the world of relapse and death due to disseminated histoplasmosis

(DH) in acquired immunodeficiency syndrome (AIDS) patients. The objective of this study is to characterise the relapse and mortality of DH in AIDS cases residents in Ceará. We performed a retrospective analysis of the medical records of AIDS patients PF2341066 who had a first episode of DH from 2002 to 2008. We analysed the outcomes until December 31, 2010. A total of 145 patients participated in the study. The mean clinical follow-up duration was 3.38 years (SD = 2.2; 95% CI = 3.01–3.75). The majority of the subjects were male with a mean age of 35 years (SD = 2.2; 95% CI = 3.01–3.75) and were born in the capital of Ceará. DH was the first manifestation of AIDS in 59% of the patients. The relapse rate was 23.3%, with a disseminated presentation

in 90% of these patients. The overall mortality during the study period was 30.2%. The majority of patients who relapsed or died had irregular treatment with antifungals or highly active antiretroviral therapy and did not have active Osimertinib price clinical follow-up. High rates of recurrence and mortality were found in AIDS-associated DH in this area of the country. “
“Invasive fungal infections are a major cause of morbidity and mortality in immunocompromised children

and premature neonates. The new class of echinocandin lipopeptides offers alternative options for treatment and prevention through a distinct mechanism of action, broad spectrum antifungal activity against Candida and Aspergillus spp., linear pharmacokinetics, few relevant drug–drug interactions and excellent tolerance. Micafungin has been the first echinocandin approved in Europe for the use in children of all age groups, including preterm neonates. Its favourable safety profile and documented clinical efficacy in all paediatric age groups make it an attractive choice for treatment of candidemia and other forms of invasive candidiasis and for prophylaxis of Candida infections in haematopoietic stem cell transplant and severely neutropenic patients. This article reviews the clinical development of micafungin and provides an update on pharmacokinetics, safety and dosing of the compound in paediatric age groups.

Lymphocytes were extracted from whole blood samples of 16 young healthy donors (28 ± 7 years,

five female and 11 male). Exclusion criteria for these donors were a history of cancer, rheumatic diseases, acute and chronic Dasatinib infections, cartilage injury and OA. The study protocol was approved by the ethics committee of the University of Heidelberg, Germany. Both patients and blood donors provided informed consent. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000. Mononuclear cells (MNCs) were isolated from bone marrow samples by Ficoll Paque plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. MNCs were then resuspended in culture medium at a density of 5 × 105 cells/cm2 (= 2·5 × 106 cells/ml). Culture medium contained Dulbecco’s modified Eagle’s medium low glucose (DMEM-LG; Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 1% penicillin/streptomycin

(Invitrogen). The cells were cultured in 175 cm2 cell culture flasks (Nunc, Roskilde, Denmark) at 37°C with 6% CO2 in a humidified atmosphere. After 24 h, with the first media exchange, non-adherent cells were discarded; afterwards, medium replacement was carried out every 72 h until the cells reached an 80% confluent layer. The cells were then detached with trypsin selleck compound (Biochrom), washed with complete medium and counted (trypan blue 0·4%; Sigma-Aldrich, Steinheim, Germany). Afterwards, MSCs were replated and cultured under the conditions described above until reaching confluence at passage 2. The ability of MSC to differentiate into chondrogenic,

adipogenic and osteogenic lineages was demonstrated according to protocols described previously [32]. MSCs were allogeneic to the lymphocytes in all co-culture experiments. Peripheral blood mononuclear cells (PBMC) were collected from whole blood samples using Ficoll Paque plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. PBMC were then separated into a mixture of CD4+CD25– and CD4+CD25+CD127– cells using magnetic separation (CD4+CD25+CD127dim/– regulatory T cell Isolation Kit II, LS and LD columns, MidiMACSTM separator, all from Miltenyi Biotec, Bergisch Gladbach, Germany). The Pyruvate dehydrogenase isolated cells were then analysed for CD4, CD25, CD127 and forkhead box protein 3 (FoxP3) (see below). MSCs derived from bone marrow (B-MSCs) and synovium (S-MSCs) from 18 patients were co-cultured with CD4+ T cells enriched in Tregs for 5 days in DMEM-LG (Invitrogen) supplemented with 10% FCS (Biochrom) and 1% penicillin/streptomycin (Invitrogen). The cells were resuspended in 48-well plates, each well containing 1 ml of medium and cells in various concentrations: T cells/MSCs 4:3 (37 500 T cells/cm2 and 28 125 MSCs/cm2), 2:1 (37 500 T cells/cm2 and 18 750 MSCs/cm2) and 4:1 (37 500 T cells/cm2 and 9375 MSCs/cm2).