Rats

were anaesthetized by inhalation of ether in air and killed by decapitation. Skin around the lower abdomen was removed, and a small incision was made on the abdominal muscle to allow insertion of a trocar. Ten ml of lavage solution (D-Hanks) was administered through the trocar into the rat peritoneum. The rat peritoneum was massaged for 2 min, and the lavage solution was retrieved by a transfer pipette into a 15-ml conical tube. After centrifugation at 450 g for 5 min, the cell pellet was resuspended Tamoxifen purchase in 5 ml PBS for staining, while the supernatant was collected and stored at −80 °C for measurement of cytokines and histamine. Typical mast cells in rat small intestine tissue or peritoneal lavage solution (RPLS) were stained with toluidine blue stain as previously described [18]. Briefly, 200 μl peritoneal

lavage fluids was dried in the air on cromolyn sodium–pretreated slides and then covered with several drops of staining solution (toluidine blue stain dissolved in 70% ethanol). After 90 s, the staining solutions were washed away quickly with the running tap water, and the stained cells were examined and counted under light microscope (Olympus, Japan). The BN rats were sacrificed after anaesthetized by inhalation of ether in air. Rat peritoneal mast cells (RPMCs) were obtained by peritoneal lavage and purified by density gradient fractionation as described previously [19, 20]. Isolated RPMCs preparations contained >98% mast cells and at least 98% of these cells were viable as checked by metachromatic staining learn more in 0.05% toluidine blue. The cells were then used for the following RT-PCR, Western blot, Ca2+ image and immunofluorescence experiments of SOCs subunits. For a mechanistic study, in vitro allergic model was re-established as follows: isolated RPMCs from control group were cultured at density of 2 × 106 cells per well for 30 min in 24-well Baricitinib tissue culture plates with

DMEM (GIBCO) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. Then, the cells were divided into control, OVA, Wortmannin (Sigma, USA) and Ebselen (Sigma, USA) group. The cells in Wortmannin group were pretreated with 100 nm Wortmannin for 15 min, while Ebselen group were pretreated with 100 μm Ebselen for 30 min. After that, all the groups of cell, except control group, were sensitized by 30% RPLS (diluted by DMEM) from OVA-treated rats for 6 h. All the cells were then challenged by 10 μg/ml OVA for 1 h and used for the following experiments of RT-PCR, Western blot and Ca2+ imaging of SOCs subunits. Intracellular Ca2+ signal was measured as described previously with minor modification [21]. Rat peritoneal mast cells (RPMCs) were incubated with 5 μm Ca2+ fluorescent probe fluo-4 AM (Invitrogen, CA, USA) for 30 min at room temperature.

Both patients were treated with a liposomal preparation of AmB and early partial resection of the infected structures followed by prolonged posaconazole maintenance therapy. Despite incomplete resection, this treatment regimen resulted in a favourable outcome in both patients, including survival of more than 17 months in one patient at last follow up.

For patients in whom complete resection of pulmonary zygomycosis is not possible, subtotal resection and treatment with liposomal AmB followed by therapy with posaconazole may be an effective treatment option. “
“The objective of this study was to Selleck Birinapant compare the antifungal activity of terbinafine (TERB) with that of lanoconazole (LAN). Test isolates, which were clinical isolates of Japanese origin, included 10 strains each of Trichophyton rubrum, T. mentagrophytes and Epidermophyton floccosum. The minimum inhibitory concentration (MIC) of TERB and LAN against each dermatophyte isolate was determined according to the selleckchem Clinical and Laboratory Standards Institute microbroth methodology, M38-A2. Minimum fungicidal

concentrations were determined by subculturing the contents of each visibly clear well from the MIC assay for colony count. All LAN MICs were ≤0.008 μg ml−1, while the TERB range was 0.008–0.03 μg ml−1. Moreover, by standard definition, LAN was fungistatic against most strains, whereas TERB was fungicidal. Both LAN and TERB demonstrated potent antifungal activity against dermatophytes; however, the lack of fungicidal activity by LAN needs to be evaluated in terms of potential clinical efficacy. “
“Cryptococcus neoformans is an encapsulated yeast-like fungus that causes life-threatening infections, particularly in immunocompromised patients. The formation of brown pigment on many media described in the literature, such as that in Niger seed (Guizotia abyssinica) agar, has been used to identify C. neoformans. The present study compares melanin production by clinical and environmental isolates of C. neoformans and other medically important yeast on two new media, Pinus halepensis

seed (PHS) agar and blackberry (BlaB) agar, and the classic medium Niger seed agar. Results obtained after the culture of 46 strains of C. neoformans, for 4, 24 and 48 h at 37 °C on these three media, showed 5-Fluoracil purchase that at 24 h, 100% of strains were pigmented on BlaB agar, 91.3% on PHS agar but only 34.8% on Niger seed agar. In conclusion, PHS and BlaB agar are two interesting new media for the rapid identification of C. neoformans isolates. “
“Invasive aspergillosis is one of the most severe complications after liver transplantation characterised by early dissemination of disease and high mortality. Recent data show that the prognosis is diminishing even further when Aspergillus terreus, a strain resistant to standard treatment with amphotericin, is isolated.

IL1-β was inhibited to its baseline in several, but not all experiments; IL-6 inhibition was always significant but almost never total. As indicated above, IL-10 secretion followed IL-1β and IL-6 secretion. We were surprised to observe that IL-10 was only mildly inhibited relative to IL-1β, and even to IL-6 (Fig. 3C). Inhibition of IL-1β was 75–100% in most experiments, and IL-6 was inhibited 40–70%, whereas IL-10 inhibition was only 20–35%. Thus, interaction with iC3b-opsonized apoptotic cells not only markedly inhibited the proinflammatory response to zymosan, but also changed the relative secretion of cytokines,

favoring a high anti-inflammatory-proinflammatory cytokine secretion ratio. Similar results were obtained while using LPS. IL-1β and IL-6 secretion were reduced from 160±25 to 31±14 and from 1820±188 to 555±88 pg/mL respectively, following one h exposure to opsonized apoptotic cells (p<0.001). In the same manner, click here interaction with iC3b-opsonized apoptotic cells downregulated MHC class II, CD86, CD83, CD40, and CCR7, as well IL-12 secretion (data not shown, see 5). In order to further find more verify that the decrease in proinflammatory cytokines was not due to decreased ingestion of zymosan, we documented zymosan uptake following

macrophage−apoptotic cell interaction. As shown in Fig. 3D, zymosan uptake was not inhibited following apoptotic cell interaction, and was even augmented. Thus, the inhibitory pattern seen in secretion of proinflammatory cytokines was not due to decreased phagocytosis of zymosan. The specificity of the uptake was further shown by using fibrinogen-coated plates. Zymosan induced proinflammatory cytokines from macrophages differentiated on fibronectin-coated plates,

resulting in IL-1β and IL-6 secretion reaching 197±21 and 2120±118 pg/mL, respectively. Thus, no proinflammatory cytokine inhibition was shown using fibrinogen as a ligand. Furthermore, addition of opsonized apoptotic cells reduced cytokine secretion to 29±11 and 611±48, respectively, following 1 h exposure to opsonized apoptotic cells (p<0.001 and p<0.001). Taken together, the results indicate that CD11b/CD18 and CD11c/CD18 response to ligand is specific to iC3b-opsonized apoptotic cells, and not every ligand (i.e. fibronectin) will induce the same response. Since it has been proposed that inhibition of the proinflammtory response is an autocrine/paracrine process 2, 4, and L-gulonolactone oxidase we were able to detect IL-10 secretion, we examined the effect of anti-IL-10. As shown in Fig. 4A and B, anti-IL-10 had no effect, suggesting an alternative mode of inhibition. Although TGF-β was not detected, anti-TGF-β was also examined because TGF-β is sometimes hard to detect and can be released in the preformed mode. As shown in Fig. 4A and B, anti-TGF-β did not have any effect on inhibition. Thus, in this system we were not able to demonstrate a paracrine/autocrine effect of IL-10 or TGF-β that led to inhibition of the immune response to zymosan and LPS.

Tolerosomes are physiologically produced as a response to dietary peptides; it is already known that enterocytes posses the molecular mechanisms for processing peptides in a similar manner to lymphocytes. The fate of tolerosomes is not precisely known, but it seems that they merge with intestinal dendritic cells, conveying to them the information that orally administered peptides must be interpreted as tolerogens. SEA can stimulate this mechanism, PLX4032 order thus favoring the development of tolerance to peptides/proteins administered subsequently via the oral route. This characteristic of SEA might be useful in therapy for regulating immune responses. The present

paper reviews the current status of research regarding the impact of SEA on the enteric immune system and its potential use in the treatment of allergic and autoimmune diseases. Staphylococcal enterotoxin A belongs to the family of staphylococcal enterotoxins, a group of molecules which have drawn the attention of researchers in the field of immunity for over 30 years. The first SE discovered was SEA, in 1966,

followed by another eight (B-E, G-J). The original observations were connected with the ability of these enterotoxins to induce toxic shock when food contaminated with Staphylococcus aureus strains was ingested (1). From the beginning, it was observed that SEs are active in very small amounts (micrograms), and are very stable. Generally, selleck products foods contaminated by them retain their toxicity after boiling or freezing. Even in the digestive tract, these proteins are not degraded by local proteases and can therefore still exert their specific actions (2). In the case of SEA, at approximately 4 hr after the ingestion of less than 1 μg, symptoms such as nausea, vomiting, and abdominal cramps appear (3). This is accompanied

by an inflammatory infiltrate abundant in PMNs in the lamina propria and epithelium of the intestinal wall. PMNs release large quantities of mediators such as histamine, leukotrienes, Parvulin and intestinal neuropeptides including substance P, all of which contribute to the clinical picture (4). The proof for the inflammatory etiology of the symptom of emesis in toxic shock is that this symptom is reversed by the administration of antihistamines. In some animal models, it has been proved that SEA also induces secretion of monocyte chemo-attractant protein 1 (5), IL-6 and IL-8 by the intestinal myofibroblasts (6). Under the influence of SEA, the serotonin concentration increases in the intestinal wall, stimulating local vagal receptors, an absolutely necessary step in the development of the gastrointestinal symptoms (7). In addition to their toxic activity, SEs stimulate adaptive immunity as SAs, which means that the number of T cells activated by these toxins is much greater than in the case of normal antigens.

Family meetings are usually a good way to interact with the indigenous patient and the family. Effective communication skills selleck compound are needed to have effective discussions. Here the clinician needs to actively listen and give time for replies and questions. Patients and families should not feel unduly pressured to choose or embark on a particular pathway of care. It can be helpful to let the caregivers know that this is a medical recommendation and that the physician is, with their assent, primarily

responsible for the decisions. Above all it should be a shared decision making process with the patient’s best interest the primary consideration at all times. It is important to discuss cultural requirements and preferences early in the conservative management see more pathway so that the impact of family and kinship relatives can be managed. Family/kinship rules may mean that certain family members of an indigenous person, who in mainstream society would be regarded as distant relatives, may have strong cultural responsibilities to that person. It is imperative therefore to identify early in the planning stages

who is the culturally appropriate person, or persons to be involved in the decision making process so that they can give consent for treatment and discuss goals of care. Where English is not the main language of the person and/or their

family, interactions and family meetings will always need to be held in the presence of a cultural broker (aboriginal liaison officer) and or an interpreter to explain treatment pathways and care issues so that informed choices are made. Informed choices can be only made Adenosine in an environment where all stakeholders can participate freely. An interpreter or translator can be an invaluable resource in such situations to ensure that information is conveyed and received accurately. The use of a family member as an interpreter may not always be appropriate and the health care team should be sensitive to these issues. Given the remoteness and accessibility issues in the life of indigenous Australians, it may be sometimes difficult to bring the patients to the ‘tertiary services’. In many instances, ‘services’ may have to be taken to the patient. One effective way of doing this is by tele or video case-conferencing with the local clinic, DMO/primary GP, patient and family as well as the renal team in attendance. The range of environmental and social conditions in the remote setting may also necessitate flexible models of care and creative solutions to sourcing equipment and medications etc. Patients in the ‘remote setting’ who have chosen the non-dialysis pathway will have to be supported and cared for at home.

5%), whereas

only one out of 27 strains isolated in Japan belonged to classical serotypes, though this strain (O142:H6) was isolated from someone who had traveled to the Philippines. The strains which were isolated in Japan were distributed in O153 and O157 serogroups. There were no common serotypes between those from Thailand and Japan. We previously ROCK inhibitor reported 5 HMA-bfpA types (34). In this study, we identified a new type, HMA-bfpA type 6 (Fig. 1). All the strains of this type were isolates from Thailand (Table 2). Most strains isolated in Japan were bfpA types 1, 4 and 5, while, those isolated in Thailand were bfpA type 2, 3 and 6. Several serotypes could be assigned to each bfpA type. The perA genes were classified as 8 HMA-types (Table 2). Most strains isolated in Japan were perA types A and B, whereas those isolated in Thailand were perA types C to H. Although perA variation was more complex than bfpA variation, each perA genotype corresponded

to a main bfpA type. Amplicons of the bfpA gene (including new HMA-type) and perA gene were sequenced. PCR amplification was performed with whole coding region primers (Table 1). Figure PLX4032 research buy 5 shows the phylogenetic tree of the perA sequences of our strains and those reported by Lacher et al. (29). The perA genotypes were clustered into four major groups, α, β, γ and δ, as described (29). Most of the isolates from Japan were in the β cluster. In this study, the new perA sequence types, β3.2, β3.3 and β3.4 were identified (Fig. 2). HMA typing produced similar results

to those of sequence typing in the polymorphism analysis on bfpA and perA. All except 4 strains showed autoaggregation (Table 2). Since aggregates of various sizes were observed, we defined the extent of autoaggregation according ID-8 to 4 categories (+++ to –) (Fig. 3b). Those in category +++ (n= 30) were huge aggregates clearly visible with the naked eye, category ++ (n = 4) aggregates of medium thickness, and category + (n= 17) small, weak aggregates (Fig. 3b). Particle measurements were also carried out on the autoaggregates in each category and a different peak was observed for each one (Fig. 3a). When morphological changes were investigated by scanning electron microscopy, we observed microcolony structures at 3 hr post inoculation. Microcolonies in category +++ were intricately intertwined, whereas in category +, they were barely visible (Fig. 3c). The rate of aggregation was quantitated by measuring the turbidity with reference to the E2348/69 strain using the representative strain of each category (Fig. 3e). Significant differences were observed among categories (P < 0.02). Adherence to HEp-2 cells has been used to identify EPEC (5, 38). In this regard, LA is a qualitative adherence pattern consisting of compact microcolonies on the surface of epithelial cells.

We conclude that cellular differentiation of pre-BI cells to a pre-BII-like stage, induced by the removal of IL-7, is delayed, but not inhibited by the doxycycline-induced overexpression

of Myc and Pim1, as judged by the retarded loss of c-kit expression, the retarded loss of clonability on stromal cells in the presence of IL-7 and by the slower gain of CD25. Furthermore, Selleckchem IWR1 acquisition of IgM on the surface or intracellularly is blocked. It appears that the Myc-single and the Pim1/Myc-double-transduced cells are arrested in differentiation before sIgM+ immature B cells. Transplantation of Pim1/Myc-double-overexpressing pre-BI cells in doxycycline-fed Rag1−/− recipient mice (Fig. 3) led to a marked expansion of CD19+ B-lineage cells in Cabozantinib mw vivo. In two separate experiments, the transplanted pre-B cells were kept either for 4 weeks (Fig. 3A–C) or for 8 weeks (Fig. 3D) in doxycycline-fed mice, followed each by a 4-week period without doxycycline in the drinking water. At 4 weeks, high numbers of transplanted cells overexpressing Pim1 and Myc were detected in BM, spleen and

peritoneum. At 8 weeks, the transplanted pre-B cells could also be detected in the swollen lymph nodes of the animals (data not shown). FACS analysis of the phenotypes of B lineage cells showed that spleens of doxycycline-induced mice, which harbored Pim1/Myc overexpressing B cells contained 100-fold higher numbers of pre-B cells, up to 6-fold higher numbers of immature IgM+ B cells, and up to twice the numbers of mature B cells than spleens of doxycycline-uninduced mice (Fig. 3B and C). The expanded number of cells detected after 8 weeks in BM, spleen, peritoneum and lymph nodes in the presence of doxycycline were, in majority, CD93+IgM− pre-B

cells (data not shown). Removal of doxycycline from the drinking water from transplanted mice 4 or 8 weeks after transplantation resulted in the disappearance of the previously expanded numbers of pre-B-, immature, and the slightly increased numbers of mature B cells from the spleen to normal numbers seen in uninduced mice (Fig. 3A, B and D). In a separate experiment, the capacities of Pim1/Myc-overexpressing pre-B cells to proliferate ex enough vivo after expansion in vivo were tested (Fig. 3E and F). These Pim1/Myc-overexpressing IgM− pre-B cells isolated from spleen and LNs of mice fed for 8 weeks with doxycycline could be propagated in vitro without IL-7 and OP9 cells in the presence, but not in the absence of doxycycline. Upon removal of doxycycline from these ex vivo cultures, the cells terminated proliferation and acquired IgM on their surface (Fig. 3F). The reasons for this oncogene-dependent inhibition of IgM expression are presently under detailed investigation.

The PBMCs from patients with TM (n = 35), patients with TH (n = 30), patients with NT (n = 21) and HC (n = 32) were examined for the subset population, defined as the percentage of Th17 cells among total CD4+ T cells using flow cytometry. Summarized

data from all individuals indicated that the proportion of Th17 cells in TM group was significantly higher than those in HC group (1.49 ± 0.59% versus 0.99 ± 0.12%, P < 0.05) (Fig. 1A,B). There was no significant difference in the frequency of Th17 cells between TH group (1.38 ± 0.42%), NT group (1.08 ± 0.52%) and HC group (P > 0.05). There was also no significant difference in the frequency of Th17 cells between TM group and TH group (P > 0.05). We also compared the number of the Treg cells in PBMCs in patients with MG to that in healthy subjects. The proportion of Treg cells in TM group (3.23 ± 0.64%) was lower than those in TH group (5.87 ± 0.51%, P < 0.05), NT group (6.27 ± 0.51%, P < 0.05) GSK126 concentration and HC group (6.21 ± 0.12%, P < 0.05) (Fig. 1C). There was no significant difference in the BGJ398 manufacturer frequency of Treg cells between TH group, NT group and HC group (P > 0.05). The results suggested that increased number of Th17 cells and decreased number of Treg cells specifically correlate with MG patients with TM but

not all patients with MG. To further evaluate possible alterations in the expression of pro-Th17 genes in MG, we tested its mRNA levels in patients with MG and healthy subjects by using real-time quantitative PCR. The values were calculated as copy numbers of interesting genes in terms of house-keeping gene (β-actin). The relative quantification values (RQ values) of mRNA are shown in Fig. 2. The expression levels of IL-17 mRNA (23.1 ± 4.7) were upregulated significantly versus those in HC group (13.8 ± 3.0, P < 0.01). Adenosine As IL-1β, IL-6 and IL-23 were involved in the generation of human Th17 cells, we further detected their mRNA expression. The expression levels of IL-1β mRNA significantly

increased in TM group (7.3 ± 2.1) versus those in HC group (4.8 ± 1.6, P < 0.05). The expression levels of IL-6 mRNA increased in TM group (8.4 ± 1.9) versus those in HC group (4.9 ± 1.3, P < 0.05). The expression levels of IL-23 mRNA in TM group (18.4 ± 2.1) increased significantly versus those in HC group (11.3 ± 2.9, P < 0.05). No differences in expression levels of TGF-β1 mRNA were found (P > 0.05). We used ELISA to detect the Th17-related cytokine levels in serum. As shown in Fig. 3, the mean concentration of IL-17A was upregulated significantly in TM group (30.0 ± 7.2 pg/ml) versus HC group (20.0 ± 4.9 pg/ml, P < 0.05). Serum levels of IL-23 were always increased in TM group (208.0 ± 85.6 pg/ml) versus HC group (93 ± 48.3 pg/ml, P < 0.01). The expression of IL-1β in TM group (72.0 ± 34.5 pg/ml) and in TH group (86.0 ± 30.1 pg/ml) increased significantly versus those in HC group (45 ± 25.3 pg/ml, P < 0.05).

On this basis, the selective killing of M2 macrophages by RAPA is not unexpected. In fact, we previously reported that in resting human monocytes, cell activation through three different signal pathways prevents death resulting from RAPA treatment: SCH727965 mouse GM-CSF/IL-3 receptors, TLR4 and IL-1β/TNF-α/IFN-γ receptors.[28]

As levels of IL-3, IFN-γ, IL1-β and TNF-α are all significantly higher in M1 than in M2 polarization, this can explain the M1 resistance to RAPA induced apoptosis. M1-polarized macrophages mediate resistance to intracellular pathogens and tissue destruction whereas M2-polarized cells are generally oriented to tissue remodelling and repair.[42] The target of RAPA action is the inhibition of mTOR, so our findings propose that the mTOR pathway is essential

in Ku-0059436 clinical trial M2 but not in M1 macrophage survival. The mTOR acts as a central sensor for nutrient/energy availability[10] and it could provide an important homeostatic mechanism for controlling the number and the function of M1 and M2 macrophages in a manner dependent upon basal nutritional status. On this basis, we can speculate that in the presence of sufficient nutrients and energy, mTOR could relay a permissive signal for M2 survival, facilitating events that drive tissue remodelling and repair. On the other hand, in conditions of limited nutrient availability, as mimicked by RAPA treatment, mTOR could preferentially ‘sacrifice’ the M2 compartment, so preserving the resistance to pathogens due to the existence of mTOR-independent pathways that regulate M1 survival at the site of inflammation. Consistent with this hypothesis is the finding that RAPA treatment impairs wound healing in patients.[43] Moreover the relevance in regulating M2 survival could add a further explanation to activity of RAPA against cancer[44] and atherosclerosis development,[45] two diseases supported also by the presence of alternative activated macrophages.[46-48] In accordance with this, Chen et al.[49] recently reported that the Vorinostat mw mTOR pathway is a critical element in the regulation of monocyte differentiation

to tumour-associated macrophages and that inhibition of mTOR by RAPA reduced tumour growth both in vitro and in vivo by modulating macrophage polarization. Beyond the impact on M2 survival, RAPA induced relevant modification of macrophage phenotype and cytokine/chemokine secretion in vitro. M1 macrophages appeared more affected than M2 and, as a general trend, RAPA unbalanced the system to classic activation. In fact, in M1 macrophages, RAPA increased the expression of CD86 and CCR7 and induced a significantly higher release of IL-6, TNF-α and IL-1β (markers of classic activation) while reducing the expression of CD206 and CD209 and the release of IL-10, VEGF and CCL18 (markers of alternative activation).

As argued by Aslin and Newport (2012), the degree of generalization is a function of the patterning of the input to which the learner is exposed. Even canonical ACP-196 mw statistical-learning studies that only test exemplars drawn from the specific stimulus materials to which the learner is exposed can be viewed as an inference problem (Goldwater, Griffiths, & Johnson, 2009).

For example, the words and part-words used as test items in Saffran et al. (1996) were drawn from the continuous stream of syllables presented during the familiarization phase. Thus, neither of these test items were exact replicas of what had been presented for “learning”. Yet, infants readily showed reliable differences in “recognition” of these test items. Thus, the proper way to conceptualize any learning task is to ask what are the most plausible inferences that the learner could make based on the patterning of the input. Reeder, Newport, and Aslin (2013) provided extensive evidence that adults will either generalize freely or restrict generalization depending on the patterning of the context in which nonsense words are presented across a family of utterances. Their task consisted of listening to several hundred utterances of variable word lengths and then being tested on (1) a subset of these familiar utterances, (2)

a set of novel utterances that conformed to the underlying grammar, and (3) a set of novel utterances that violated the underlying grammar. find more Crucially, the number of grammatical categories and which nonsense words were assigned to these categories were unknown to the subjects. In each of eight separate experiments, the patterning of the nonsense words that surrounded a critical target category differed—in some experiments all possible surrounding contexts were presented in Akt inhibitor the familiarization utterances, in others some of the surrounding contexts were consistently absent, and in yet others

only a single context was present. Thus, as in Gerken (2006), the surrounding contexts varied from providing consistent evidence for generalization to inconsistent evidence for generalization, and finally little or no evidence for generalization (i.e., strong evidence for restricting generalization). Moreover, in two follow-up experiments that more closely mimicked the variability in word frequency (K. D. Schuler, P. A. Reeder, E. L. Newport, & R. N. Aslin, unpublished data) and the presence of subcategories (Reeder, Newport, & Aslin, 2010) that add a further level of context, adults readily generalized or restricted generalization depending on these same principles of patterning in the surrounding contexts. Thus, distributional cues are sufficient to induce learning and modulate generalization.