We also thank Sunao Iyoda (National Institute of

We also thank Sunao Iyoda (National Institute of ABT-199 ic50 Infectious Diseases: NIID) and Yan Lu (NIID) for assistance with HEp-2 adherence assay, and Shizuko Ichinose (Tokyo Medical and Dental University) for assistance with the electron microscopy.

Hidemasa Izumiya (NIID) kindly provided the EPEC reference strain. This study was supported by grants-in-aid for Food and Chemical Safety from the Ministry of Health, Labor, and Welfare of Japan. “
“Many differences exist between human immature and mature natural killer (NK) cells, but their respective molecular signatures and transcriptional regulators are relatively unknown. To gain new insights into the diversity and developmental regulation of human NK cells, we used data from high-resolution microarrays with independent verification to describe a comprehensive comparative analysis between immature decidual NK (idNK) cells with a CD56brightCD16−T-bet− phenotype and mature peripheral NK (mpNK) cells with a CD56dimCD16+T-bet+ phenotype. This study shows that many novel growth factors, cytokines, and chemokines are expressed by NK cells, and they may regulate NK-cell development or function in an autocrine manner. Notably, we present that idNK and selleck kinase inhibitor mpNK cells are enriched

for homeobox and zinc-finger transcription factors (TFs), respectively. Additionally, many novel candidate transcriptional regulators are common to both idNK and mpNK cells. We further describe the transcriptional regulatory networks of NK cells and show that the endogenous growth factors, cytokines, and TFs enriched in idNK cells regulate each other and may contribute to idNK-cell immaturity. 5-Fluoracil Together, these findings provide novel molecular signatures for immature and mature NK cells, and the novel candidate regulators identified here can be used to describe and further understand NK-cell differentiation and function. “
“Tumour necrosis factor-α-induced

protein-8 like-2 (TIPE2) is a newly identified immune negative regulator. The abnormal expression of TIPE2 has been found in several human inflammatory diseases. However, the expression level and clinical significance of TIPE2 in childhood asthma remain unclear. In this study, we detected TIPE2 expression in peripheral blood mononuclear cells (PBMC) from 42 children with asthma and 39 healthy controls by RT-PCR, qRT-PCR and Western blot. We also detected the levels of serum total immunoglobulin E (IgE), eosinophil (EO), interleukin-4 (IL-4) and interferon-γ (IFN-γ) and analysed the correlations of TIPE2 expression with IgE, EO, IL-4 and IFN-γ. The results showed that TIPE2 mRNA and protein expression were decreased in children with asthma compared with healthy controls. The levels of IgE, EO and IL-4 in the children with asthma were obviously higher than those in normal controls, while the level of IFN-γ in patients with asthma was significantly lower than that in healthy subjects.

For the 0 1-μg dose, lymphocyte and eosinophil numbers were signi

For the 0.1-μg dose, lymphocyte and eosinophil numbers were significantly higher in 20- compared with 1-week-old mice (* in Fig. 3A, B). For the 10-μg dose, this was opposite; the cell numbers decreased with age (Fig. 3A, B). In a separate study, mice were sensitized by i.n. instillation of OVA in Al(OH)3 and challenged i.n. with OVA. The main and interaction effects are reported above the figures. When a significant effect of age or a significant sex and age interaction

was found, the result of the post hoc test is given on the figure. Fig. 4A shows the OVA-specific IgE response in 1-, 6- and 20-week-old female and male mice. Significant main effects of both sex and age were found. Sensitized females produced higher levels of OVA-specific IgE compared with males (Fig. 4A). GS1101 Further, the IgE response increased with age as 20-week-old mice had significantly higher levels than 1-week-old mice. The same pattern was observed for OVA-specific IgG1 production; females had significantly higher antibody production than males, and the response in both sexes increased with age (Fig. 4B). Cells from both SLNs and MLNs were stimulated with OVA ex

vivo. In MLNs, IL-4 was undetectable. Only IL-10 secretion was influenced by the sex of the mice, with females releasing significantly more IL-10 than males (Fig. 5A). IL-5 and IL-13 secretion was higher in 1-week-old mice compared with Selleckchem GSK3 inhibitor older mice (Fig. 5B, C). INFγ was affected by age in the same manner as IL-17A secretion (Fig. 5D, E); 6-week-old mice had significantly lower IFNγ and IL-17A secretion than until 20-week-old mice and for IFNγ also significantly

lower than the 1-week-old mice. A significant age and sex interaction was found for the total number of cells in MLNs (Fig. 5F). The post hoc test revealed that only in the oldest age group did females have significantly higher number of cells compared with males (bracket in Fig. 5F). In SLNs, IL-4, -5, -10, -13 and IFNγ were undetectable and IL-17A produced at very low levels (data not shown and Fig. 5G). IL-17A production increased with age but was not affected by sex. The total number of cells in SLNs was unaffected by both sex and age (Fig. 5H). Control groups of mice were immunized i.n. with OVA alone. When comparing the OVA and OVA + Al(OH)3 treatments, MLN cell numbers, but not SLN cell numbers, were highly increased after using the adjuvant for sensitization, and this was observed for all age groups (data not shown). In contrast to the control groups (data not shown), a pronounced airway inflammatory cell influx dominated by macrophages, lymphocytes, some epithelial cell shedding and in particular by eosinophils was found in BALF of the mice. However, only lymphocytes, epithelial cells and eosinophils were significantly affected by the investigated experimental factors. The number of lymphocytes, eosinophils and epithelial cells in BALF was significantly higher in female mice compared with male mice (Fig. 6A, B, C).

In WT mice, the number of total thymocytes reached its peak betwe

In WT mice, the number of total thymocytes reached its peak between 2 and 8 wk of age (Fig. 1A). The total number of thymocytes from LAR−/− mice at corresponding ages was slightly lower than from WT mice. As shown in Fig. 1B, the average number of total thymocytes in LAR−/− mice was significantly lower than in WT mice. After 11 wk, the number of total thymocytes was similar in both LAR−/− and WT mice (Fig. 1A and B). We then investigated the effect of LAR deficiency on thymocyte differentiation by analyzing CD4 and CD8 expression. The most immature thymocytes

do not express Gefitinib in vitro CD4 or CD8. Immature thymocytes then differentiate into CD4+CD8+ DP thymocytes while passing through a transient CD4−CD8+ (CD8SP) differentiation stage 20. After positive

selection, they lose either CD4 or CD8 expression and differentiate into CD8SP or CD4+CD8− (CD4SP) mature thymocytes. To examine the effects of LAR deficiency on thymocyte differentiation, we analyzed the expression of CD4 and CD8 on thymocytes from WT mice and LAR−/− mice by flow cytometry and calculated the percentage of different thymocyte subpopulations. Of the total thymocytes, 4.0±1.3% and 2.5±0.6% were DN in LAR−/− and WT mice, respectively (Fig. 2), and 84.5±1.2% and 86.3±2.0% were DP, respectively. Furthermore, 8.2±1.4% and 8.5±1.4% of the total thymocytes LY2157299 were CD4SP in LAR−/− and WT mice, respectively, while 3.2±0.4% and 2.7±0.5% were CD8SP in LAR−/− and WT mice, respectively. Taken together, the percentage of DN thymocytes was higher (p=0.0011), that of DP thymocytes was lower (p=0.0022)

and that of CD8SP thymocytes was cAMP higher (p=0.009) in LAR−/− mice compared with WT mice. In CD8SP thymocyte population, the percentage of CD8SP cells that expressed high level of TCRβ was decreased in LAR−/− mice compared with WT mice (p=0.04) (Supporting Information Fig. 3), whereas DP or CD4SP thymocyte population expressing high level of TCRβ was not altered significantly in WT and LAR−/− mice. The results indicate that the percentage of CD8SP cells that expressed no or low level of TCRβ, i.e. immature CD8SP thymocytes, was increased in LAR−/− mice compared with WT mice. Taken together, the differentiation of DN thymocytes to DP thymocytes via intermediate CD8SP thymocytes is partially impaired in LAR−/− mice. The differentiation stages of the DN thymocytes were further subdivided using CD44 and CD25 expression (DN1, CD44+CD25−; DN2, CD44+CD25+; DN3, CD44−CD25+; DN4, CD44−CD25−). We previously showed that IMT-1/LAR was first expressed on DN2 thymocytes and that most DN3 thymocytes continued to express IMT-1/LAR 18. Figure 3 and Supporting Information Fig. 4 show that the proportion and the number of DN subsets defined by the expression of CD44 and CD25 on DN thymocytes was corresponding in LAR−/− and WT mice.

In particular, tissue-selective recruitment of immune cells to cu

In particular, tissue-selective recruitment of immune cells to cutaneous tissues, a complex multistep cascade mediated by a large variety of cytokines, chemokines, and adhesion molecules, is thought to have a pivotal role [28, 29]. Among adhesion molecules, induction of ICAM-1, a ligand for LFA-1- and Mac-1 molecules, on the surface of epidermal keratinocytes contributes to infiltration and retention of T-cell populations in the skin, and has been proposed as an important regulator

in skin immune reactions [30]. In this regard, we found that the reduced expression of ICAM-1 in PS-5-treated keratinocytes resulted in impaired adhesiveness of T cells Navitoclax molecular weight to IFN-γ-activated keratinocytes in an in vitro cell-contact model. T-cell recruitment in inflamed skin tissue is also due to the release of a set of proinflammatory chemokines, including CXCL10 and CCL2, by cytokine-activated Everolimus cell line keratinocytes [4, 31]. In line with this knowledge, in this study, we demonstrated that the migratory ability of T lymphocytes toward sups from keratinocytes pretreated with PS-5 and activated by IFN-γ is drastically reduced compared with that observed in supernatants from control cells. Finally, we confirmed the antiinflammatory

action of PS-5 on IFN-γ signaling by an ex vivo approach based on the use of ROS1 IFN-γ-activated explants of human skin treated with PS-5 mimetic and compared to those treated with

control peptide. We found that, other than inhibiting STAT1 phosphorylation in the epidermis of organ cultures of normal human skin, PS-5 peptide impaired the epidermal expression of the inflammatory ICAM-1 and HLA-DR membrane molecules, as well as that of the CXCL10 chemokine, corroborating the effectiveness of this SOCS1 mimetic peptide in reducing the inflammatory responses elicited by IFN-γ-activated human keratinocytes. Increasing evidence suggests that JAK proteins might be a viable target for immunosuppressive drugs against psoriasis and other immune-mediated skin diseases, and the design of potent and selective JAK2 chemical inhibitors could be crucial for the development of optimized therapeutics with minimal adverse physiological effects [32, 33]. On the other hand, limited information concerning the use of peptido-mimetics in inflammatory skin diseases, including psoriasis, is available, likely due to the short-term in vivo stability of these molecules. In this regard, a unique demonstration of the effectiveness of the topical application of antiangiogenic peptides based on pigment epithelium-derived factor in improving psoriasis exists [34].

MiRs are small (20–22 nucleotide) non-coding RNAs that degrade or

MiRs are small (20–22 nucleotide) non-coding RNAs that degrade or inhibit translation of mRNA by binding to recognition

sequences on the mRNA sequence. One miR can modulate a number of genes and as such function as a master regulator. In the case of apoptosis signalling for instance, several miRs have been shown to imprint an apoptosis-resistant phenotype on tumour cells. Several miRs have been reported to modulate apoptotic signalling by TRAIL and other TNF family members. In GBM, a specific miR (miR21) has been reported as highly overexpressed in >90% of tumours analysed. Interestingly, inhibition of miR21 significantly blocked GBM outgrowth, while co-treatment of anti-miR21 therapy with neural stem cells expressing sTRAIL resulted in synergistic inhibition of tumour growth in vivo. An important consideration to make regarding all of these combinatorial strategies is the possible I-BET-762 price sensitization of normal cells. For instance, synergistic pro-apoptotic anti-cancer activity upon combination Pirfenidone of sTRAIL with proteasome inhibition yielded a therapeutic window in hepatoma cells, but was also associated with enhanced toxicity towards hepatocytes [71]. In addition, hepatocytes were strongly sensitized to Fas upon initial priming with TRAIL [72]. Hepatocytes indeed appear the most TRAIL-sensitive type of cell, with aggregated TRAIL preparations strongly reducing hepatocyte

viability [73]. Therefore, it is apparent that purely homogenous sTRAIL as well as the rational design of non-toxic combinatorial strategies is required for effective anti-cancer strategy in humans. From a conceptual point of view, the efficacy of sTRAIL is likely to be hampered by several factors, including rapid clearance from Nitroxoline the circulation by the kidney. Indeed, sTRAIL has an approximate

half-life of only 30 min in primates and a similar pharmacokinetic profile in humans in a phase I clinical trial [32,74]. Together with the ubiquitous expression of TRAIL receptors in the human body this may severely limit tumour accretion. Moreover, many tumours express higher levels of TRAIL-R2 compared with TRAIL-R1, whereas TRAIL-R2 signalling is only poorly activated by sTRAIL [75]. We and others have attempted to overcome these drawbacks by fusing sTRAIL to an antibody derivative, such as an antibody fragment. The resultant trimeric molecule will be ∼180 kDa and likely has a longer circulation half-life, as renal clearance should be impeded at these higher molecular weights. The antibody targeting domain of the fusion protein will ensure better tumour accretion and retention (for schematic see Figure 4) [76–80]. Importantly, antibody fragment-mediated binding to a cell surface-expressed target antigen converts the sTRAIL into membrane-bound TRAIL that efficiently signals apoptosis via TRAIL-R1 but also TRAIL-R2 in a mono- and/or bi/multi-cellular manner [81,82].


Within compound screening assay this inflammatory area, a minimum of six images (fields) were collected. Image analysis and processing were performed with LSMix (Zeiss) or LaserSharp, Confocal Assistant, Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA) and Image Tool software (UTHSCSA, San Antonio, TX, USA). Analyses were performed by counting the total number of cells in six to nine fields acquired and calculating the average cell number per field for each patient. This procedure was performed for each parameter analysed, allowing determination

of the total number of inflammatory cells (total number of DAPI+ cells within the inflammatory infiltrate), the number of FITC (TCR Vβ regions) or PE-Cy5 (CD4+) single-positive cells, and the number of double-positive cells. The counts were performed blindly for each parameter for each patient. The results are representative this website of two experiments per patient. Statistical analysis was performed as indicated in each figure legend. For comparison of means between control versus CL, individual Student’s t-tests were used for each given Vβ-expressing population. For comparison of specific

Vβ-expressing CD4+ T cell populations between media alone and SLA, paired Student’s t-tests were used. For comparison of the percentage of cells within each Vβ population expressing a given marker (CD45RO, cytokines, etc.), the data were treated with the Tukey–Kramer analysis of variance (anova) test within the jmp statistical package (SAS Institute Inc., Cary, NC, USA). All correlation analyses were performed using Spearman’s correlation coefficient contained within the jmp statistical package (SAS Institute Inc.) and reported with its associated r2 and P-value. The clinical characteristics of the 12 patients with CL used in this study are shown in Table 1. All patients were from an endemic area near Salvador, Brazil (see Materials and methods) and participated in the study after informed consent through the donation

of peripheral blood. Regardless of participation in the study, all patients received medical care. The patient ages ranged between 14 and 50 years (mean 25·08 ± 11·15) and time of lesion, as reported by the patient, ranged from 8 to 120 days at time the blood was taken and measurements were made. The total area measured of ulcers varied from 12 to 4-Aminobutyrate aminotransferase 272 mm2 (mean 151·44 ± 103·87). All patients presented with positive Leishmania skin tests (MST), while measurements existed for 11 patients, ranging from 72 to 910 mm2 (mean 329·72 mm2 ± 229·66). We performed a comparative analysis of the frequency of T cells expressing given Vβ regions 2, 5·1, 5·2, 8, 11, 12, 17 and 24 from CL and from non-infected individuals. The mean frequency of cells expressing Vβ 5·2 and 24 was increased slightly in the actively infected CL group compared to the non-infected control group (P = 0·006 and P = 0·02, respectively) (Fig. 2).

73 m2 or kidney disease

at hospitalization) did not have

73 m2 or kidney disease

at hospitalization) did not have albuminuria (ACR ≥ 30 mg/g).8 Cross-sectional studies in people with type 2 diabetes and microalbuminuria have generally shown GFR to be normal, however, increased GFR (hyperfiltration) have been observed. For example in a Danish study 158 microalbuminuric patients had an increased GFR of 139 ±  29 mL/min compared with 39 normoalbuminuric patients (115 ± 19 mL/min) and 20 control subjects without diabetes (111 ± 23 mL/min).9 However, the cross-sectional study by Premaratne et al.10 of 662 Australian people with type 2 diabetes showed no significant difference in AER and prevalence of microalbuminuria between hyperfilters and normofilters. Although not recognized PD0325901 cost as a stage of CKD, hyperfiltration (GFR > 130 mL/min

per 1.73 m2) represents an early phase of kidney dysfunction in diabetes. However, its clinical significance remains controversial. By definition, this phase can only be detected by measurement of GFR. In people who do not have diabetes, the expected rate of decline in GFR with ageing is approximately 1 mL/min per year.11 A proportion see more of people with type 2 diabetes show a more rapid decline in GFR, in the absence of microalbuminuria or macroalbuminuria.12 In people with type 2 diabetes and established nephropathy, some but not all longitudinal studies have documented a decline in GFR without

intervention of about 10 mL/min per year.13 In people with type 1 diabetes, and overt kidney disease, the extent of early reduction in AER GNE-0877 by ACEi predicts the degree of protection from subsequent decline in GFR).14 Whether this occurs in people with type 2 diabetes is not yet known. Lack of uniformity in results on decline in GFR in longitudinal studies is in part due to study design, since most studies have focussed on albuminuria and have been too short to document clinically significant changes in GFR. In a Japanese study over 48 months, no change in GFR was demonstrated in 48 patients who were either untreated or treated with nifedipine, enalapril or both drugs.15 In another study of 103 normotensive Indians over 5 years, there was no change in GFR during treatment with placebo or enalapril.16 By contrast, two studies have shown a significant decline in GFR in at least one study arm. In a 5 year study of 94 middle aged normotensive Israelis, GFR remained stable in those treated with enalapril but declined in those treated with placebo.17 This study used the inverse of the serum creatinine level as an index of GFR. In a 3-year study of 18 hypertensive Italians, the GFR (measured isotopicaly) decreased in those treated with cilazapril or amlodipine.

The objective of this study was to describe cryptococcosis mortal

The objective of this study was to describe cryptococcosis mortality and associated medical conditions in the US for the period 2000–2010. Cryptococcosis-related deaths were identified from the national multiple-cause-of-death dataset. Mortality trends and comparison analyses were performed on overall cases of cryptococcosis and by subset [i.e. clinical manifestations of disease and human immunodeficiency virus (HIV) status]. A matched

case–control analysis was also conducted to describe the associations between this disease and comorbid medical conditions. A total of 3210 cryptococcosis-related deaths were identified. Cerebral cryptococcosis was the most commonly reported clinical manifestation of the disease. Approximately one-fifth of the decedents (n = 616) had a co-diagnosis of HIV. Mortality rates were Cell Cycle inhibitor highest among men, blacks, Hispanics, Native Americans and older adults. Poisson regression analysis indicated a 6.52% annual decrease in mortality rates for the study period. HIV (MOR = 35.55, 95% CI 27.95–45.22) and leukaemia (MOR = 16.10, 95% CI 11.24–23.06) were highly associated with cryptococcosis-related deaths. Cryptococcosis mortality declined significantly during 2000–2010. However, the disease continues to cause appreciable mortality in the US. With the majority of decedents having no HIV co-diagnosis, there is still

much to be learned about the epidemiology of this mycosis. “
“Numerous studies have suggested a link between fungal sensitisation MAPK Inhibitor high throughput screening and severity of asthma. However, few studies have specifically evaluated the relationship between Aspergillus sensitisation and asthma severity. This study was aimed at investigating the clinical significance of Aspergillus sensitisation in asthma. In this prospective cross-sectional study, patients with asthma were subjected to pulmonary function test and an intradermal Aspergillus skin test (AST) apart from a GPX6 detailed clinical history and physical examination. Assessment of asthma

severity was carried according to the Global Initiative for Asthma (GINA) recommendations, Asthma Control Test (ACT) and the mini Asthma Quality of Life Questionnaire (mini AQLQ). Based on AST, the cases were dichotomised into Aspergillus-sensitive and AST-negative groups. There were 417 (193 males, 224 females; mean age, 34 years) asthmatic patients of whom 219 (52.5%) showed Aspergillus sensitisation. The severity of disease as per the GINA criteria and the dose of ICS required for asthma control were similar in the two groups. The Aspergillus-sensitive group had poorer pulmonary function than the AST-negative group [AST positive vs. negative: percentage predicted mean (SD) forced expiratory volume in the first second : 73.1(23.8) vs. 77.9(22.7), P = 0.04; mean (SD) FEV1/forced vital capacity (FVC) ratio: 68.2(13.3) vs. 74.3(15.7), P = 0.0001]. The mini AQLQ scores were similar in the two groups.

[44] On the other hand, very aggressive EAE induction (for exampl

[44] On the other hand, very aggressive EAE induction (for example, repeated immunization with high dosages of heat-killed Mtb) completely abrogates IFN-β efficacy Cobimetinib in wild-type mice (Inoue et al., unpublished data). Hence, EAE induced by moderately aggressive immunization may develop as a mixture of two EAE subtypes; NLRP3 inflammasome-dependent and -independent. When two subtypes of EAE are ongoing, it may be possible that IFN-β efficacy correlates with levels

of NLRP3 inflammasome dependency in EAE development. Although two subtypes of EAE may be occurring simultaneously within some of the disease in WT mice, the findings are summarized as follows: NLRP3 inflammasome-dependent EAE is a disease that responds to IFN-β treatment, whereas NLRP3 inflammasome-independent EAE is a disease that is resistant to IFN-β (Fig. 2). Previous studies have shown that passive EAE induced by Th17 cell transfer is resistant to IFN-β treatment, whereas the disease induced by Th1 cells responds to IFN-β treatment.[81] The result is counterintuitive because IFN-β inhibits Th17 responses;[62, 65] and it will be of great interest to understand why Th17-mediated EAE cannot be treated by IFN-β. Activation status of the NLRP3 inflammasome is not known in the Th17-mediated EAE model, but the result (resistance of Th17-mediated passive EAE to IFN-β) does not conflict with IFN-β resistance in NLRP3 inflammasome-independent

EAE. This is because the Th17 response itself is not the reason

for NLRP3 inflammasome-dependent EAE progression.[44] Further studies will be necessary to determine whether or not these two types BGB324 of IFN-β-resistant EAE (Th17-type EAE and NLRP3 inflammasome-independent Cell press EAE) share the same mechanism. It is currently unknown whether NLRP3 inflammasome-independent MS exists. It is also not known what type of event is an equivalent of ‘aggressive immunization’ in MS. However, if the current findings on the correlation between NLRP3 inflammasome activation and response to IFN-β in EAE can be applied to MS, it might be possible to predict MS patients who do not respond well to IFN-β therapy. For example, the activation status of the NLRP3 inflammasome might be a prediction marker. Or, it might be possible to identify prediction markers by screening molecules that show altered expression in NLRP3 inflammasome-independent EAE. It is also possible to test such molecules for prognosis markers, or even as molecular targets of selected treatment(s). “
“Human Vγ9Vδ2 T cells play a crucial role in early immune response to intracellular pathogens. Their number is drastically increased in the peripheral blood of patients during the acute phase of brucellosis. In vitro, Vγ9Vδ2 T cells exhibit strong cytolytic activity against Brucella-infected cells and impair intracellular growth of Brucella suis in autologous macrophages.

Before the initiation of the

study, the animals were test

Before the initiation of the

study, the animals were tested for S. dysenteriae 1 and S. flexneri 2a infections by ELISA against lipopolysaccharides of test pathogens. Institutional animal ethical committee granted approval to conduct this study. The invasive ability of the strains was confirmed using the guinea-pig keratoconjunctivitis test (Sereny, 1955). The conjunctival sac of one eye of each guinea-pig was inoculated with 109 CFU of the test strain and CP-673451 clinical trial observed for the development of keratoconjunctivitis after 1–3 days. Forty guinea-pigs were assigned randomly to four groups, each group with 10 animals. For the determination of an effective infectious dose with and without cecal tie-up, 28 guinea-pigs were divided randomly into seven groups, each with four animals. In addition, 32 guinea-pigs were used in the immunological studies in four groups, each with eight animals. Of these, two groups each were used for immunization with heat-killed S. dysenteriae 1 (NT4907) and S. flexneri 2a (B294) to evaluate the protective efficacy and the rest served as controls. All JQ1 datasheet the experiments were performed twice. In order to determine the infectious dose in a luminal model, six different doses (106, 107, 108, 109, 1010 and 1011 CFU mL−1) of the reference strain S. flexneri 2a (2457T) were experimented. After finding the required dose that confers significant signs of bacillary

dysentery, two different strains of HSP90 Shigella using guinea-pigs were tested. The test animal was sedated by an intramuscular injection of a mixture of ketamine (35 mg kg−1 body weight, Sterfil Laboratories Pvt Ltd, India) and xylazine (5 mg kg−1 body weight, AstraZeneca Pharma India Ltd, India). The cecum was brought out through a 3 cm

midline incision without compromising the blood supply. A permanent cecal tie was made 4 cm apart from the ileocecal junction so that the ligation completely obstructed the cecal lumen above this junction while maintaining the ileo–ceco–colic connection (Fig. 1). The purpose of this ligation was to prevent the entry of cecal contents into the proximal colon and disruption of water absorption. During the surgery, hydration of the exposed intestine was maintained with sterile PBS. At the cecocolic junction, 1 mL of test inoculum was injected into the lumen of the colon. The colon was placed back inside the abdominal cavity and the incision was closed. The incision site was checked twice a day for signs of infection, and each time, it was washed with a 1% chlorhexidine solution (Saatman et al., 1986) soaked with sterile gauze pads during the next 72 h. We did not find any wound infection in any of the guinea-pigs during the postsurgical period. After the surgery, the animals were allowed to consume food and water and were observed for the development of shigellosis for 48 h.