The RD1 and RD9 genomic regions are present in all virulent and clinical strains of M. tuberculosis but deleted in all M. bovis BCG vaccine strains [29]. The importance of proteins encoded

by RD1, both for diagnosis and vaccine development, is highly suggestive because three genes of this region have been conclusively shown to be expressed in M. tuberculosis and are major antigens recognized by antibodies (ORF14) and T cells (EsxA and EsxB) of patients with TB and/or healthy but exposed individuals [30, 31]. In particular, EsxA and EsxB proteins are recognized by T cells with protective phenotype, i.e. memory and effector IFN-γ secreting T cells in mice and human T cells producing large quantities of IFN-γ [32–35]. To prepare DNA vaccine constructs using the plasmids, DNA fragments corresponding to PE35, PPE68, EsxA, EsxB and EsxV genes were PCR amplified by using gene-specific primers, and Decitabine cell line the amplified DNA were first cloned into pGEM-T Easy vector, before subcloning into

pUMVC6 and pUMVC7. This was performed to facilitate the cloning of appropriate DNA into the eukaryotic expression vectors, as has been performed previously for prokaryotic expression vectors [14, 15]. A total of 10 recombinant DNA plasmids (five for each vector) were constructed. All the aforementioned recombinant DNA plasmids were studied for expression and immunogenicity of the cloned fragments by immunizing mice. The results show that both the vectors induced antigen-specific cellular proliferation to PE35, EsxA, EsxB, and EsxV proteins. However, recombinant pUMCV6 induced relatively better responses learn more than recombinant pUMCV7.

The improved responses with recombinant pUMCV6 suggest that hIL2 secretory protein acted as a better MAPK inhibitor adjuvant and enhanced cellular immune responses, assessed by antigen-induced proliferation of splenocytes, to the fused mycobacterial proteins more effectively than the tPA signal peptide. The relevance of antigen-specific cellular proliferation induced by DNA vaccine constructs with protection against M. tuberculosis challenge has been demonstrated in the mouse model of TB [36]. Although the results of this study, showing induction of antigen-specific immune responses to the antigens encoded by genes present in DNA vaccine constructs, are interesting, the work should be extended to demonstrate their protective efficacy in challenge experiments with M. tuberculosis using various animal models of TB, e.g. mice, guinea pigs, rabbits and monkeys. If found effective in animals, such vaccines may be useful in both prophylactic and therapeutic applications in humans. Furthermore, DNA-based vaccines expressing M. tuberculosis-specific antigens may even be useful in BCG-vaccinated subjects as preventive vaccines, because revaccination with BCG has not shown beneficial effects [4, 37, 38] and may even be combined with BCG to improve its protective efficacy [39].

Retrospectively alignments of the investigated hUTY-peptides with those of canine-, murine- and rat-sequences (Table 3) revealed conservation of K1234 in all four species. For W248, the most immunogenic peptide in our setting (positive

in 3 dogs) changes only appeared in 0-2 AAs which had no influence on peptide-sequence/properties and their immunogenic potential. Furthermore, buy Alectinib W248-data could already be shown in mice [55]. Highest divergence was determined for T368, with the human- and canine-peptides being similar at most. As determined in this work for UTY, substantial homologies and conservation of immune-reactivity, functionality, proteins, peptides (including MHC-presentation) and isoforms were already described for canines in comparison to

humans, cats, mice, rats, apes and cows by others in vitro and in vivo [30, 56-68]. Further in vitro-culture experiments of lymphocytes from in vivo immunized females with DLA-identical-male cells should be performed to strengthen our preliminary data of our first proof-of-principle experiments. Furthermore, higher response of in vivo T cell proliferations might be exhibited by peptide-loaded (single-peptides or peptide-mix/pool) Y-27632 cost male-DCs or male-PBMCs, as well as investigating human- and canine-UTY-peptides in parallel. Thereby, using human- and canine-UTX-homologue peptides, unspecific X-chromosomally derived reactivity can be excluded and the DLA-binding efficacy of the human/canine-UTY/UTX peptides will be verified as well. Non-hematopoietic-cells (fibroblasts, keratinocytes) should be examined with respect to their target cell function as well as their UTY-expression profiles. In further studies we want to transfer our setting in clinical settings, especially in a context of stem-cell transplantation or T cell transfer for treatment Montelukast Sodium of human leukemia: Normally, UTY is not restricted to cells of hematopoietic origin, but the level of expression may differ in various tissues. Adoptive immunotherapy with Y-chromosome-encoded UTY would be feasible in certain circumstances. This first proof-of-principle experiment should demonstrate that hUTY-peptides are presented on male-canine cell-surfaces triggering

a male-specific immune-response. Interpretation of our experiments could be enhanced by cloning some canine-T cells via limiting-dilution-culture recognizing one of the three hUTY-derived-peptides, permitting more detailed examinations of the antigenic specificity and functional properties of CD8+ as well as CD4+cells [43]. Adoptive immunotherapy with DLT after SCT provides a potent strategy to curatively treat haematological malignancies [69]. However, the use of DLT is limited by occurrence of GvHD and sometimes by the poor-response of the patients [70]. Optimized sensitization of donor T cells against antigens presented by leukemic cells could improve DLT. Therefore, ex vivo CTL-generation against UTY for the treatment of recurrent leukemia is reasonable.

There were no major complications, and very satisfactory results have been obtained.

This retrospective study showed that both options of raising a large DIEAP flap for unilateral breast reconstruction, namely unipedicled flap based on large medial perforator/s plus additional venous discharge or double-pedicle flap, are safe. Preoperative examination of the dominant perforator/s with CDS and/or MDCT is mandatory in both cases. © 2010 Wiley-Liss, Inc. Microsurgery 2010. “
“Breast conservation surgery in the treatment of early stage breast cancer has become increasingly utilized as a means to avoiding mastectomy. While partial mastectomy defects (PMDs) may often be cosmetically acceptable, some cases warrant consideration of reconstructive options, and while several reconstructive options have been described in this role, a series of deep Wnt inhibitor inferior epigastric perforator (DIEP) flaps Selleck MAPK Inhibitor Library has not been reported to date. A cohort of 18 patients undergoing PMD reconstruction with a DIEP flap were included. Patient-specific data, operation details, cosmetic results, and complication rates were assessed. Oncologic outcomes, in particular recurrence rates, were also evaluated. In our series there were no cases of partial or total flap necrosis, and overall complications were

low. There were two cases of wound infection (both had undergone radiotherapy), managed conservatively, and one case of reoperation due to hematoma. There were no cancer recurrences or effect on oncologic management. Cosmetic outcome was rated as high by both patients and Progesterone surgeon. The results were thus comparable with other reconstructive options. Although autologous reconstruction has an established complication rate, our results suggest that the DIEP flap may be of considerable value for delayed reconstruction of selected larger partial mastectomy defects. © 2010 Wiley-Liss, Inc. Microsurgery, 2011. “
“The latissimus dorsi (LD) muscle flap is one of the most versatile flaps used for reconstruction of soft tissue defects. With knowledge of its anatomy, harvest of the segmental LD muscle has been introduced as a reliable technique with

the advantage of muscle preservation. We devised a new harvest technique for the segmental LD flap using a limited transverse incision to elevate a less bulky distal segment of the muscle with a sufficient pedicle length obtained by intramuscular dissection of the vascular pedicle. Two cases, in which this technique was effectively applied to reconstruct plantar defects after wide excision of malignant melanoma with a maximally efficient use of donor and recipient tissues, are presented. Satisfactory results were gained with stability in walking. When the defect size permits use of a segmental muscle and the long pedicle is needed, this pedicle-lengthened segmental LD muscle harvest technique would be a valuable method. © 2013 Wiley Periodicals, Inc. Microsurgery 33:491–495, 2013.

After 12 weeks of medication, the IPP group showed persistently high storage symptoms than the non-IPP group. Conclusion: BPH patients with IPP showed less improvement of storage symptoms after 12 weeks of medication. This study suggests that

IPP may be a possible cause of intractable storage symptoms in early treatment. “
“Objectives: Intravesical injection of onabotulinumtoxinA (i.e. Botox) provides effective treatment for overactive bladder. However, treatment-related adverse events (AEs) remain problems. This study investigated the effect of AEs after onabotulinumtoxinA injection BIBW2992 datasheet on the success rate for idiopathic detrusor overactivity (IDO). Methods: A total of 174 patients who received the first single intravesical onabotulinumtoxinA 100U injection for refractory IDO were included. The onabotulinumtoxinA related AEs including acute urinary retention (AUR), large postvoid residual (PVR, ≥150 mL), difficult urination, urinary tract infection, gross hematuria and general weakness were recorded. The success rate was determined based on patient perception of bladder condition improved by two scales. The short-term (3 months) and long-term (up to 24 months) success rates were analyzed

according to the occurrence of these AEs. Results: A successful outcome was reported by 138 (79.3%) patients at 3 months. AUR occurred in 12 (6.9%) patients, large PVR developed in 81 (46.6%) and 73 (42%) needed straining to void. Gross hematuria occurred in 17 (9.8%) patients, urinary tract infection developed in 27 (15.5%) and general weakness was noted in 6 (3.4%). The occurrence of AUR did not affect the therapeutic this website results. Patients having large PVR and difficult urination had a significantly higher success rate at 3 months. Long-term success rates up to 24 months showed no significant difference between patients with and without AEs. Conclusions: AEs after intravesical

100U onabotulinumtoxinA Arachidonate 15-lipoxygenase for IDO were frequently encountered. However, the occurrence of AUR, large PVR or difficult urination did not affect the final therapeutic outcome. “
“Objectives: To determine if rat bladders augmented with an acellular Japanese bovine pericardium-derived biomaterial (CardioDISC [CD]) could support bladder reconstruction, and increase bladder volume and compliance. Methods: Female Sprague–Dawley rats were randomly divided into three groups (n = 5 each). After partial cystectomy, bladders were closed without augmentation (non-augmentation) or augmented with porcine small intestinal submucosa (SIS) or CD, both of which are acellular. At 1, 2, 4 and 8 weeks after surgery, bladder volume and compliance were measured. The bladders were then analyzed by immunohistochemistry for smooth muscle actin (SMA), urothelium uroplakin III (UPIII), and nerve fiber S100. Results: At 4 weeks after augmentation, the SMA-positive cells from the host bladder tissues were present near the regions augmented with CD.

The vast majority (62%) had abrupt PD technique failure. This is a marked difference to dated reports of AVF use after concurrent PD and AVF formation. It raises the possibility that the formation of back-up fistula may be another method to reduce the need for vascular catheter use. “
“Automated peritoneal dialysis (APD) and double-bag continuous ambulatory peritoneal dialysis (CAPD) are the two current standard modalities of peritoneal dialysis (PD). Outcomes

of these two modalities have not been well described. A single-centre, retrospective review was carried out to compare the treatment failure rate of APD and double-bag CAPD. Treatment failure was a combined endpoint including death and technique failure. Cox regression was used to compare risk (hazard ratio, HR) of Selleck Ibrutinib treatment failure in APD and CAPD. There were 121 patients included in this study, 55 with APD and 66 with CAPD. APD patients had significantly lower risk of treatment failure (death and technique failure)

than CAPD patients (HR 0.58, 95% confidence interval [CI]: 0.37–0.91, P = 0.02). The lower risk of treatment failure in APD compared to CAPD was mainly caused by the significantly lower risk of technique failure (HR 0.30, 95%CI: 0.10–0.93, P = 0.04). The mortality rates of the two modalities were not significantly different (HR 0.69, 95%CI: 0.42–1.12, P = 0.13). Our data suggest Deforolimus cost that APD may have lower risk of treatment failure compared with double-bag CAPD. These potential benefits of APD might justify the use of this modality despite its higher cost. “
“Aim:  This pilot study compared mycophenolate mofetil (MMF) and tacrolimus (Tac) in the treatment of severe membranous lupus nephritis (MLN). Method:  This was a 24 month prospective, randomized, open-label multi-centre exploratory study on Chinese patients with biopsy-proven pure Class V MLN with nephrotic syndrome. Patients were randomized to treatment

with either MMF or Tac, both in combination with prednisolone and the efficacy and tolerability outcomes were examined. Results:  Sixteen patients were included, seven in the MMF and nine in the Tac treatment arm. At 24 months the complete response, partial response and overall response rates were 57.1% vs. 11.1% (P = 0.049), 14.3% vs. 44.4% (P = 0.197) and 71.4% vs. 55.6% (P = 0.515) in the MMF and Tac groups, respectively. The two groups had similar reduction of proteinuria and BCKDHB longitudinal profiles of serum albumin and creatinine levels. Serum creatinine remained stable in both groups, except in two patients who had a transient increase associated with high Tac blood levels. Adverse events in the MMF group included herpes zoster in one patient and reversible leucopenia in another, while in the Tac group four patients had severe infections and one developed new onset diabetes. No relapse occurred during the study period. Conclusion:  Both MMF and Tac when combined with corticosteroids are effective treatment options for severe MLN.

A statistical test based on measures of central tendency comparison was not applicable to the particular case of anti-IgM combined with IL-21. A P-value less than 0·05 was considered statistically significant. B cells die from apoptosis if maintained unstimulated in culture [31]. After 3 days, spontaneous apoptosis was higher in CD27+ than in CD27– B cells (79·2 versus 57·6%, P < 0·001) (Fig. 2a). When B cells are stimulated, they are rescued from apoptosis.

The effectiveness of the rescue depends upon both the kind of stimulus used and the subpopulation of B cells. For CD27– B cells, the strongest rescue effect was induced by anti-CD40 followed by CpG-ODN and to a lesser extent by anti-IgM, whereas for CD27+ B cells, CpG-ODN appeared to be the strongest rescue stimulus (Fig. 2b). Nevertheless, all the stimuli evaluated were more efficient in the CD27– than in the CD27+ LY294002 research buy population: anti-CD40 (77·9 versus 23·9%, P < 0·001), CpG-ODN (71·4 versus selleck kinase inhibitor 57·3%, P < 0·01) and anti-IgM (52·7 versus 36·9%; P < 0·01) (Fig. 2b). Proliferation was evaluated simultaneously. Anti-CD40 and anti-IgM did not induce proliferation of either CD27– or CD27+ B cells while CpG-ODN induced proliferation of both subpopulations (Table 2). Although CpG-ODN

induced a lower level of proliferation on CD27– than CD27+ B cells (PI = 0·1 versus PI = 1·8, respectively; P < 0·001) (Table 2), it induced higher rescue from apoptosis in the CD27– population (Fig. 2b). These aforementioned results suggest that proliferation and rescue from apoptosis are two independent processes. CD27– B cells from CVID MB0 patients were less sensitive to rescue from apoptosis when stimulated with a T-dependent stimulus (anti-CD40) than control subjects (65·4 versus 77·9%, P < 0·05)

(Fig. 3a). They were also less sensitive to rescue from apoptosis when stimulated with a T-independent stimulus (CpG-ODN) than control subjects or CVID MB1 patients, although differences did not reach statistical significance (58·8 versus 71·4 and 63·0%, respectively, P = 0·075). CD27– B cells from CVID MB1 patients were rescued from apoptosis similarly to controls, regardless of the stimulus used (Fig. 3a). After BCR engagement with anti-IgM CD27– B cells from both CVID MB0 and MB1, patients very were rescued equally from apoptosis than healthy controls. CD27+ B cells from CVID MB0 patients, stimulated with either a T-dependent (anti-CD40) or a T-independent stimulus (CpG-ODN), were less sensitive to apoptosis rescue than control subjects (6·0 versus 23·9%, P < 0·01; and 23·2 versus 57·3%, P < 0·05, respectively) and CVID MB1 patients (6·0 versus 30·6%, P < 0·001; and 23·2 versus 65·7%, P < 0·01, respectively). They were also less sensitive to rescue from apoptosis after BCR engagement with anti-IgM than control subjects (19·2 versus 36·9%, P < 0·05) or CVID MB1 patients (19·2 versus 38·2%, P < 0·01) (Fig. 3b).

In this regard, specific non-pathogenic IgM aabs [14, 15] right throughout life [16] play a major role in assisting the complement dependent removal of cellular breakdown products by phagocytic cells [17–19]. Such immune elimination of cellular waste prevents possible chemical modification of self components, thereby preventing an autoimmune disease causing pathogenic aab response [20]. Inappropriate presentation

of exogenous and endogenous ag can cause serious chronic illnesses. The disorders resulting from exogenous and endogenous ag–derived mishaps are generally alleviated or treated by medication, often with limited success. Yet it has long been anticipated that a vaccination technique, one that was not merely prophylactic but rather could be administered ex post selleck chemicals llc facto, could function, by the appropriate presentation of ag, to terminate such disorders. As far as exogenous ag are concerned, their presentation in a live form, e.g. as components of virulent bacteria,

MAPK inhibitor can set up a serious illness in a host. Endogenous ag, likewise, when presented in modified form, e.g. modified by drugs or other chemicals, can set up (by invoking the development of pathogenic aabs) autoimmune diseases characterized by serious injury to organs and associated functional disturbances [12, 21–27]. If cancer cell–surface residing cancer-specific ag are weakly antigenic (not recognized as abnormal self) then the cancer will establish itself, spread and be life-threatening. Inappropriate presentation of disease causing exogenous and endogenous ag begs the question: how can we prevent or treat chronic ailments (such as cancer, autoimmune diseases and chronic infections) specifically and without causing side effects? The presentation of an exogenous ag, as it is foreign to the host, will in every instance evoke an immune response – initially

a primary, and then, if the host has already had contact with the ag, a secondary immune response. In most instances the immune response will involve IgG abs in eliminating/neutralizing the invading organism and its products. By eliminating the ag, homeostasis is re-established. Prophylactic vaccinations, Flavopiridol (Alvocidib) effective against various invasive microscopic life forms, can prevent the occurance of serious illnesses by priming the immune system to react quickly against such potential invaders. Through the systematic introduction of bacteria and viruses in inactive or attenuated forms, prophylactic vaccination programmes have resulted in the control/elimination of many exogenous ag from our external environments that previously caused harm (e.g. small pox, polio, rabies, diphtheria, tetanus, measles, etc.). Ag presentation (i.e. by vaccination) up until now has not been able to deal with endogenous ag–induced disorders.

Membranes were then subjected to incubation with AP conjugated to goat anti-rabbit IgG (Bio-Rad), washed, and finally developed using the AP Conjugate Substrate Kit (Bio-Rad). The multiplier of the highest dilution of the sample that, when visually assessed, gave an apparently positive reaction was defined as the amount of M protein. Finally, the amount of M protein in each sample was expressed as the mean of the results obtained

in assays performed in triplicate. For example, when a sample showed the highest positive reaction on 23 of the 2-fold dilutions (21, 22, 23, 24, and so on) of the original sample, the tentative amount of M protein was defined to be the exponential component 3 of the multiplier,

23. Statistical analyses of the data, Erlotinib mw including ANOVA, were carried out using GraphPad Prism version 4.03 (GraphPad software). Differences were considered statistically significant if the P value was <0.05. The DNA fragments of csrRS, including their open reading frame and flanking regions, were amplified through PCR using Pyrobest DNA polymerase. PCR was conducted under the following conditions: 94°C for 5 min, followed by 30 cycles each consisting of 94°C for 30 s, 45°C for 30 s and 72°C for 3 min, and finally 72°C for 7 min. The primers csrR-n3 and csrS-c5 were used for the PCR reaction. The following primers were used for sequencing: csrR-n4; csrR-n6; csrS-n2; csrS-n4; csrS-c4; csrS-c6; csrS-c7 and csrS-c10. The primers mga-c5 and Proteases inhibitor mga-n3 were used to amplify the mga gene and the flanking region by means of conventional PCR using Pyrobest DNA polymerase. The following primers were used in the sequence analysis: mga-c5; mga-n3; mga-c1; mga-c4; mga-n1 and mga-n2. Each PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Acquisition of the sequence data was entrusted to Takara Bio. The primers for the sequencing are listed in Table 1. Streptococcus pyogenes was grown in 5 mL of BHIY broth for approximately 18 hr. 4.7 mL of fresh BHIY was then added to 0.3 mL of the overnight culture; because the mRNA of

the M protein is generated largely during the early logarithmic phase and then degenerates rapidly, cells in the phase (OD600 = 0.3∼0.4) were allowed to grow for ∼2.5 hr, then mixed of with 2 volumes of RNA Protect Bacterial Reagent (Qiagen) and kept at room temperature for 5 min. Total RNA was subsequently extracted using the RNeasy Protect Bacterial Mini Kit (Qiagen) according to the manufacturer’s protocol. Oligonucleotide primers and probes specific for emm and proS genes were prepared according to a previously described method (17). RT-PCR was performed using the TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems, Foster City, CA, USA). The RT-PCR mixture (50 μl) contained 25 μl of 2 ×  Master Mix without uracil N glycosylase, 1.

1c). E7AS partially and completely blocked IL-32 and COX-2 expression, respectively (Fig. 1c), suggesting that factors other than COX-2 can induce IL-32. It has been reported that a single siRNA targeting the E7 coding region should inhibit the expression of both E6 and E7 proteins simultaneously31 and so E7AS could completely block COX-2 expression. Immunohistochemical staining for both COX-2 and IL-32 revealed the co-localization of these signals in invasive primary cancerous tissues (Fig. 1d). Expressed E7 induced significant increases in the activities of both the IL-32 and COX-2 promoters. As shown in Fig. 2, the HPV-16 E7 oncogene stimulated the promoter activities of Selleckchem EPZ-6438 both IL-32 (Fig. 2a) and COX-2 (Fig. 2b)

in a variety of cervical cancer cell lines, and E7AS specifically neutralized the E7-mediated activation of both the IL-32 (−746/+25) and COX-2 (−880/+9) promoters. In Fig. 2(a), there was no significant increase of IL-32 promoter activity induced by the control IL-32p itself (without E7) in the C33A/pOPI3 control cells (data not shown). Nor was there a significant increase of COX-2 promoter activity induced by the control E7 itself (without COX-2p) in the C33A/pOPI3 cells (Fig. 2b, data not shown). To determine the mechanism underlying the HPV-16 E7-mediated stimulation of COX-2 and IL-32, COX-2 was over-expressed in SiHa and CaSki cells and IL-32

expression was evaluated with RT-PCR and Western blot analyses. The IL-32 mRNA and protein expression levels were increased by COX-2 over-expression (Fig. 3a). In addition, IL-32 and E7 expressions were reduced in a Birinapant purchase dose-dependent manner by treatment with the COX-2-specific inhibitor NS398 in SiHa and CaSki cells (Fig. 3b). The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki cells. Interleukin-32 levels were determined in the supernatants of COX-2 over-expressing and NS398-treated SiHa and CaSki cells using a sandwich IL-32 ELISA as reported nearly previously,30 and significant expression levels of IL-32 were not detected in the culture media (data not shown) compared with the intracellular expression levels of IL-32. This result

supports the notion that IL-32 would not be secreted from cells as reported previously.12,26 Collectively, these data show that COX-2 is an upstream regulatory factor of HPV-16 E7-mediated IL-32 stimulation. To assess the regulatory effects of IL-32 on the expression of COX-2 mediated by the HPV-16 E7 oncogene in cervical cancer cells, SiHa and CaSki cells were transfected with IL-32γ and IL-32 siRNA, respectively, in independent experiments. The results of RT-PCR and Western blot analyses demonstrated that E7 and COX-2 were down-regulated in cells (over-expressed with IL-32γ) over-expressing IL-32γ and recovered by IL-32 siRNA (Figs 4a and 5a). The broad band of IL-32 proteins detected by Western blotting as shown in Fig. 3(b), suggested the various expressed forms of IL-32 proteins.

In contrast, IFN-γ-mediated killing of selleck kinase inhibitor the microsporidian Encephalitozoon intestinalis in CMT-93 cells was dependent on IDO activity [61]. Hence, the ability of the host epithelial cell to generate IFN-γ-mediated antimicrobial killing mechanisms may be countered by parasite survival strategies including blockade of IFN-γ signalling. The mechanisms by which cellular innate inflammatory responses are initiated by Cryptosporidium infection are poorly understood. One possible pathway would involve TLRs expressed by immune and nonimmune cells

that are important inflammatory sensors of specific molecular structures of microbial pathogens. The TLRs in enterocytes play dual roles in protecting buy Doxorubicin the mucosal surface by helping to maintain homeostasis and promoting inflammation following mucosal injury [62]. Studies with human biliary epithelial cells (cholangiocytes) infected with C. parvum suggest that signalling though TLRs is important in the initiation of the inflammatory response of these cells.

Cholangiocytes were found to express TLRs and, significantly, infection by C. parvum attracted both TLR2 and TLR4 to the site of parasite development on the epithelial cell surface [63]. Parasite development upregulated expression of β-defensin-2 by a mechanism dependent on NF-κB activation. Depletion of TLR2, TLR4 or the TLR adaptor molecule MyD88 by iRNA blocked NF-κB activation and β-defensin expression. In addition, MyD88-deficient cells were

more susceptible to infection than normal cells [63]. These findings suggest that during C. parvum infection, elements of the epithelial inflammatory response are induced by signalling through TLRs that leads to NF-κB activation. The parasite molecules that bind to TLRs have not been identified, however. Further investigation demonstrated that TLR4 expression was increased in infected cholangiocytes and this was directly related to decreased expression of the microRNA let-71 and was NF-κB-dependent [64]. Indeed, other features of cholangiocyte immunological responsiveness to infection were regulated by different clonidine microRNAs [65]. Unfortunately, the role of TLRs in activation of intestinal epithelial cells that are most relevant to cryptosporidiosis has not been extensively investigated. However, addition of the TLR9 ligand CpG to the human intestinal epithelial cell line HCT-8 before infection with C. parvum significantly inhibited reproduction of the parasite [66]. It is not entirely clear at present how important TLRs of myeloid cells are in the development of the immune response to Cryptosporidium. A recent report suggested that sporozoite antigen-induced activation of dendritic cells to produce IL-12 may be TLR-dependent as cells from MyD88−/− mice that lack signalling for most TLRs were unresponsive to antigen [45].