Briefly, race has been shown to modify the association between bacterial vaginosis and incident STI.25 One study found that certain cytokine and chemokine single-nucleotide polymorphisms were associated with ethnicity among HIV-infected individuals. The authors hypothesized Anti-infection Compound high throughput screening that heritable variations in certain of these loci may contribute

to the acquisition or progression of HIV infection.26 Further, the concept of race is a complicated one. The National Institutes of Health has historically used self-identified racial categories. Individual patients frequently do not self-identify with one of these categories and thus are classified as ‘other’. A newer technology uses single-nucleotide polymorphisms to create families of ethnic derivation called ancestry informative markers.27 These require obtaining biologic samples and laboratory work by a reputable facility so are not used frequently. However, if race is an important component of an individual HIV risk study, consideration

can be given to collection of more detailed ethnicity data. There exists a vast body of literature detailing the association between genital tract infections and HIV acquisition MLN8237 purchase and transmission. Much recent work has focused on herpes simplex virus-2 (HSV2) given the ulcerative and inflammatory nature of the infection and the high prevalence of the infection. If having HSV2 impacts shedding of HIV and the risk of transmission, then curbing the

shedding caused by this infection alone might decrease the burden of HIV infection worldwide. Herpes simplex virus-2 has been shown to increase viral load of HIV in both plasma and the genital tract, independent of the level of immunodeficiency.28 The etiology of increased shedding of HIV in the presence of HSV appears to be immunologically mediated. Rebbapragada et al.29 termed the interaction between HSV2 and HIV-1 ‘negative mucosal synergy’. While HSV suppression appears to decrease the risk of shedding HIV among women already infected with HIV, it does not appear to protect against acquisition or transmission of HIV-1.30,31 Herpes simplex virus-2 is not the only infection that alters mucosal immune handling of HIV. A less noticed but still before highly prevalent virus that may impact on genital shedding of HIV is human cytomegalovirus (CMV). The prevalence of CMV varies by geographical location, but after infection, it establishes lifelong latency. It can reactivate or hosts can be re-infected. A group well known for their CMV expertise recently developed a cervical explant study of CMV and HIV co-infection. They found that HIV appeared to enhance CMV in co-infected tissues which produced inflammatory cytokines. This explant model may be a useful tool for future studies examining the impact of CMV on HIV expression and vice versa.32 Frequently encountered STI have also been implicated in altering mucosal immunity.

CD147 has also been linked to the regulation of T-cell development in thymus. In periphery, CD147 is expressed on activated lymphocytes especially activated regulatory T cells (Tregs) within the CD4+ FoxP3+ subset. We previously demonstrated deleterious effects of CD147 in renal inflammation caused by ischemia and renal fibrosis. As CD147 identifies activated human Tregs, the attention has become extended to the autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus. Interleukin

(IL)-17 producing T cell and Treg also serve important roles in the pathogenesis of SLE. However, the molecular mechanism involving CD147 remains unknown. We therefore investigated the role of CD147 in lupus nephritis. Methods: Lupus nephritis was induced BMN 673 mw in CD147 deficient mice (Bsg−/−) or wild-type mice (Bsg+/+) with an intraperitoneal injection of pristane (0.5 ml/each mice). They were sacrificed at 6 months after an injection for histological and biochemical analyses. Kidney, spleen and thymus were analyzed. Results: There was no difference between Bsg+/+ and Bsg−/− in

serum anti-nuclear/anti-dsDNA MG-132 price antibody during the experimental period, whereas serum C3 decreased in Bsg−/−. Mesangial and endothelial cells proliferations, macrophages and CD4+ T cells infiltration, wire loop lesion and albuminuria were prominent in Bsg−/− mice. Consistent with these data, IgG, C3 and C1q depositions in Bsg−/− glomeruli were predominantly observed. By flow cytometry analysis, no obvious difference in the number of Treg was found in both genotypes, whereas IL-17A producing CD4+ T cells (Th17) were higher in Bsg−/− spleen than Bsg+/+. Th17-related gene expressions were prominent in Bsg−/− kidney. CD4+ T cells from Bsg−/− significantly

increased IL-17A level more than Bsg+/+ under Th17-skewing conditions. Interestingly, STAT3 activation, essential for Th17 differentiation, was enhanced by lack of CD147. Treatment with agonistic anti-CD147 antibody was downregulated the STAT3 activation. Conclusion: Lack of CD147 promotes Th17 differentiation through the STAT3 activation, eventually leading to the development of lupus nephritis. IKEUCHI HIDEKAZU, HIROMURA KEIJU, TSHILELA science KADIOMBO, A, KAYAKABE KEN, SAKURAI NORIYUKI, SAKAIRI TORU, KANEKO YORIAKI, MAESHIMA AKITO, NOJIMA YOSHIHISA Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine Introduction: Recently, we reported that multitarget therapy using tacrolimus (TAC) and mycophenolate mofetil (MMF) was effective in inducing early remission and in yielding a high remission rate in patients with active class III, IV, V lupus nephritis (LN) (Mod Rheumatol, 2013). Here, we conducted a follow-up study. Methods: All 16 patients in the previous study, 2 men and 14 women, 34.3 ± 8.

7), anti-CD8β (53–5.8), anti-TCRβ (H57–597), anti-CD44 (IM7). Where required, cells were incubated with Streptavidin-allophycocyanin (BD Biosciences). Anti-CD127-(A7R34)

and control-PE mAbs were purchased from e-Bioscience (San Diego, CA, USA). Anti-CD132-(4G3) and control-PE and anti-CD122- (TM-β 1) and control-FITC mAbs were purchased from BD Biosciences. Anti-TSLP-R- and control-PE goat polyclonal anti-mouse were purchased from R&D (Minneapolis, MN, USA). Samples were analyzed by a BD FACSCantoII (BD MI-503 clinical trial Biosciences) using FACSDiva software (v. 6.1.2). Dead cells were excluded by propidium iodide (PI). In some experiments, cells were fixed in phosphate-buffered saline (PBS) containing 30% methanol and 0.4% paraformaldehyde (PFA) before flow cytometric analysis. Data were analyzed using FlowJo software (v. 8.8.6) (Tree Star, Inc., OR, USA). After membrane staining and cell fixation as above, cells were permeabilized with PBS containing 0.2% Tween 20, 1% PFA, 1% BSA, and stained with either anti-Foxo1 (C29H4) or control anti-histone H2B Ab (both from Cell Signaling Technology, Beverly, MA, USA), for 30 min on ice. Cells were washed twice and stained with goat anti-rabbit

IgG-FITC secondary Ab (Invitrogen, Life Technologies Corp., Carlsbad, CA, selleck chemicals llc USA) for 30 min on ice. After washing, cells were analyzed by flow cytometry as above. CD8+ T cells were purified (≥80% pure) by negative magnetic selection (Dynal Mouse CD8+ Negative Isolation kit, Invitrogen Life Technologies) from pooled spleens [[11]]. Percoll gradient separation (Amersham Biosciences, GE Healthcare, Piscataway, NJ, USA) was performed as described [[44]] and cells of intermediate density (55–65% interface) were those collected. This fraction contained 60–70% CD44high cells within the CD8+ T-cell population. Discarded high- and low-density fractions contained for the most part respectively viable CD44int/low cells and dead cells/cell debris with few viable CD44high cells [[44]]. Intermediate density fraction CD44high CD8+ T cells were labeled

with CFSE (Molecular Probes, Eugene, OR, USA) and injected i.v. into WT, IL-15 KO, and IL-15Rα KO B6 mice (1–1.5 × 106 cells/mouse). CD8+ T cells (≥98% pure) were obtained from either pooled spleens or pooled BM by positive magnetic selection with anti-CD8β FITC mAb and anti-FITC microbeads (Miltenyi Biotec, Auburn, CA, USA) [[11]]. From these cells, highly purified CD44high CD8+ T cells were obtained by FACS sorting with a BD FACS-Aria (BD Biosciences) and used for real-time PCR analysis [[45]]. Total RNA was extracted from T cells by TRIzol (Invitrogen Life Technologies). One microgram of total RNA was used for cDNA first-strand synthesis according to the manufacturer’s protocol for Moloney MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA).

One aim was to try to identify expert practice as applied to PID, Pembrolizumab in vitro while another was to understand the impact of experience upon such practice. We conducted a cross-sectional study among members of ESID and the AAAAI. Individuals who were full members of ESID in 2006 and members of AAAAI in 2005 were eligible for inclusion in this study. Members

of the AAAAI were included as described [5], and those of ESID who met eligibility were sent the study questionnaire with an accompanying covering letter. A close replica of the questionnaire administered to the members of the AAAAI in 2005 was designed to be self-administered via the internet [5]. The aim was to collect the specialist perspectives on therapy for PIDs from members of ESID, for comparison with the findings from members of the AAAAI. Changes made prior to distribution were only minor, related mainly to European compared to American English, as the goal was to keep the two questionnaires as similar as possible. All changes made to the survey instrument were see more approved by the ESID Board to ensure applicability to a European audience. A print format reproduction of the survey instrument is available as Appendix A and the original AAAAI survey is available as a supplement to the previous publication [5]. Some of the topics addressed in this survey instrument included utilization of IVIg for specific diseases,

dosing and frequency of IVIg administration, utility of subcutaneous immunoglobulin therapy (SCIg), use of prophylactic antibiotics and health-care concerns. A covering letter from ESID, explaining the purpose of the questionnaire, was sent via e-mail to full members of ESID, approximately 450 physicians or paediatricians with a link to a non-incentivized, web-based questionnaire. Three follow-up e-mails were sent as reminders to help increase survey participation,

which was also conducted for the AAAAI members. Responses were collected electronically from July 2006 to September 2006 in a database. Each member of ESID was allowed to respond once to PAK5 the questionnaire. Duplicate responses were identified by careful analysis of name, e-mail and location fields. These repeated responses were examined closely and if the response pattern was the same in each entry, only one entry was preserved and the rest were removed. If there were multiple responses with different response patterns, all entries from the physician were removed as there was no way to determine which entry was the desired response. The original data from the AAAAI survey were analysed again for the purposes of this paper and duplicate entries treated in the same fashion to allow for optimal comparison between the two data sources. AAAAI respondents were categorized into two groups as before [5]: a ‘focused’ group that reported that > 10% of their practice was devoted to patients with PID, and a ‘general’ group where ≤10% of their practice was devoted to patients with PID.

Twenty-four hours later, mice from each group were inoculated with either a mixture of 5 × 105 CFU B. pertussis and 5 × 105 CFU B. parapertussis (1 : 1 mix) or with 5 × 105 CFU B. parapertussis alone. The following day, mice were reinjected with the appropriate

antibody to maintain neutrophil depletion. Mice were euthanized on day 4 postinoculation, the respiratory tracts were harvested and the bacterial loads of the two Bordetella species were determined. In neutrophil-depleted mice, the competitive relationship between B. pertussis and B. parapertussis was unchanged compared with control mice (Fig. 6a). There was also no significant difference in the bacterial loads between neutrophil-depleted and control mice infected with B. parapertussis alone (Fig. 6b). From these data, we conclude that neutrophils do not play a major role in the dynamics of these two organisms in coinfection CAL-101 nmr of naïve mice, nor in B. parapertussis infection. In this study, we have demonstrated that infection with B. pertussis enhances the ability of

B. parapertussis to colonize the same host in a mixed infection and that B. parapertussis outcompetes B. pertussis. When mice were coinfected with equal numbers of B. parapertussis and B. pertussis, greater numbers of B. parapertussis were recovered from the mixed infection at the early stages and through the peak of infection. In other studies, we found that by day 21 ACP-196 postinoculation, B. parapertussis was the click here only organism recovered (data not shown). Bordetella parapertussis outcompeted B. pertussis over a range of inoculum ratios, and when B. parapertussis was the predominant species in the inoculum, B. pertussis was quickly outcompeted and almost cleared from the host at the peak of infection. Bordetella parapertussis still had an advantage when the time of inoculation was staggered, with B. pertussis, followed by B. parapertussis at a later time point, from which we conclude that competition for adherence is not the reason for the advantage of B. parapertussis. Overall, these results suggest that B. parapertussis gains an advantage over B. pertussis at the very early (but postadherence) stages

of a mixed infection in this mouse model. Our results differ from those of a recent report (Long et al., 2010), in which no advantage of B. parapertussis over B. pertussis in a mixed infection was observed, and B. parapertussis did not gain an advantage from coinfection with B. pertussis compared with a single strain infection. The reason for this difference is not clear, but may be due to the use of a different mouse strain (C57BL/6), different ages of mice (10–12 weeks), higher inoculum dose (107 CFU) or different bacterial strains (antibiotic-resistant derivatives). In our study, B. parapertussis not only outcompeted B. pertussis, but was also recovered in greater numbers than those observed in infections with B. parapertussis alone. From these observations, we hypothesized that B.

6c). The present study provides evidence for a role of the CCR3/Eotaxin pathway in local proliferation and mobilization of CD34+ cells in the airways after allergen exposure. We have determined that CD34+ CCR3+ cells increase in BM, blood and airways after allergen exposure, and further demonstrated that allergen-induced newly produced eosinophil-lineage-committed (CD34+ CCR3+ BrdU+) lung cells have the capacity to proliferate in situ after allergen exposure. Significantly, IL-5 and eotaxin-2 each alone Nutlin-3a mouse stimulated

in vitro CFUs of lung CD34+ cells but not BM CD34+ cells. Moreover, delivery of eotaxin-2 to the airways of IL-5 transgenic mice resulted in a substantial increase of CD34+ cells in BAL and in vitro transmigration assays show that BM and blood CD34+ CCR3+ cells migrate in response to eotaxin-2. These data, together with our observations showing that systemic treatment with a depleting anti-CCR3 antibody abolished

both CD34+ and Sca-1+ cells in airways to levels similar to control animals, suggest a role of this chemokine receptor in lung progenitor cells. The present study showed that allergen-sensitized and allergen-exposed animals displayed a significant increase in CD34+ CCR3+ cells (relative to allergen-sensitized but saline-exposed animals) in not only the BM, but also in blood and airways. We further demonstrate that a proportion of the CD34+ CCR3+ cells in the airways stain positively for Sca-1, which confirms that some of these cells are likely to be haematopoietic stem cells. That is, Sca-1 GSK2126458 price is considered to be a stem cell marker, and has recently been shown to be involved in regulating the repopulation ability of haematopoietic stem cells in mice.28,29 Previously it had been shown

that both immature and mature BM eosinophils express CCR3 and that the expression is higher in BM from patients with atopic asthma compared with controls, suggesting that there is an increased pool of CCR3+ immature and mature eosinophils available for rapid mobilization.14,30,31 In addition, the expression of CCR3 has been shown to be up-regulated during maturation of CD34+ cells to circulating eosinophils, suggesting a role in the trafficking of metamyelocytes to inflamed tissue.31 Furthermore, an increase Rapamycin nmr in CD34+ cells in sputum has been reported in atopic asthmatic patients as well as in nasal polyp tissue.32 The increase of CD34+ cells in the nasal mucosa of patients during a pollen season, suggests that progenitors are recruited into the local airway tissue by allergen-dependent mechanisms; here they may differentiate into more mature cells within the site of allergic inflammation (i.e. in situ haematopoiesis).13,22,33–35 These parallel phenomena in allergic mice and asthmatics imply that the mouse model has relevance to the human disease in relation to eosinophil maturation and trafficking.

No typical EEG alterations were observed. Repeated 14-3-3 assay was positive after a first negative test. Neuropathology this website showed classical CJD changes with small cortical foci of large confluent vacuoles and relatively well-preserved cerebellar cortex. The most striking feature was the presence of abundant Kuru-type plaques in both cerebral cortex and subcortical white matter. Sparse Kuru-type plaques

were also seen in cerebellum, although only in white matter. Immunohistochemistry showed, in addition to unicentric plaques, diffuse synaptic and patchy perivacuolar, as well as plaque-like and periaxonal pathological prion protein deposits (PrPres). Western blot studies demonstrated the co-occurrence of PrPres types 1 and 2 in frontal cortex and a relatively weak type 2 signal in cerebellum. PRNP genotyping revealed methionine homozygosity at codon 129 and excluded mutations. selleck products This case shows a previously undescribed combination of histopathological features which preclude its classification according to the current phenotypic and molecular sCJD classification.

The observation demonstrates that Kuru-type amyloid plaques mainly involving the cerebral white matter may also occur in sCJD cases with short clinical course and the co-existence of PrPres types 1 and 2. This case further highlights the complexity of the correlations between histopathological phenotype and PrPres isotype

in prion diseases. “
“Mycoplasma pneumoniae is a well-known cause of atypical pneumonia. CNS involvement is a relatively frequent extrapulmonary manifestation, most commonly manifesting as encephalitis in the pediatric population. We present two unusual cases Progesterone of M. pneumoniae encephalitis that presented with symptoms and imaging findings suggesting mass occupying lesions, and worsening altered mental status. Biopsy of the lesions was necessary in both cases to aid with diagnosis. Histopathologic features excluded neoplasm, and established the diagnosis of encephalitis, but did not point toward its etiology. The only finding that indicated M. pneumoniae as the most likely pathogen was serum IgM positivity in the absence of any other identifiable infectious source, and complete neurologic recovery following specific anti-mycoplasmal treatment. The patients were successfully treated with antibiotics and steroids, with the second case also requiring intravenous immunoglobulin and anti-epileptics. The clinical presentation and histopathologic findings suggested an immune-mediated pathogenesis, but acute disseminated encephalomyelitis was excluded due to extensive gray matter involvement. Disease resolution despite status epilepticus and herniation in case 2 is a novel finding of the study.

1 to 8.2%, respectively, and in corresponding infected this website mice could increase to 6.8 and 23.1%, respectively (data not shown). With the frequency of NK cells increasing with age, this could explain why the younger infected control mice survive more frequently (Fig. 5) than their older counterparts (Fig. 3), and is consistent with lung NK cells being detrimental to mice infected with high-dose influenza. Not only did antibody-mediated reduction of NK cells reduce weight loss and mortality in high-dose influenza infected mice, but adoptively transferring NK cells from influenza-infected mice also exacerbated weight loss and increased mortality in infected mice. To our knowledge, this is the first demonstration

of passage of virus-induced NK cell-orchestrated

pathology from one animal to another. Also, interestingly, transfer of NK cells from virally infected mice to naïve uninfected mice did not lead to pathology. This may imply that ongoing severe influenza infection in the host may be necessary to sustain expression of effector molecules, expression of relevant NK-cell receptors, and/or induce expression of their ligands on cells of surrounding tissue for NK cells to mediate pathology. The transfer of NK cells from uninfected control mice to virus-infected mice did not enhance weight loss or mortality. This and the preceding discussion may suggest that the Selumetinib contribution of NK cells to pathology is not strictly determined by NK-cell numbers,

but possibly whether those NK cells have been adequately exposed to and stimulated by an environment experiencing influenza infection. Our demonstration that cells expressing multiple NK-cell markers in influenza-infected lung largely display an activated phenotype with IFN-γ expression, CD107a at the cell surface, and low cell surface NKp46, is consistent with our adoptive transfer experiments, and suggests that NK cells must be activated to mediate pathology. The mechanism(s) by which NK cells are exacerbating pathology remains to be elucidated. The NK cells we recovered from lung of influenza-infected mice were mature (CD27loCD11bhi), and many appeared to display an activated Sodium butyrate phenotype. The expression of cell surface CD107a indicates recent release of cytolytic components including granzymes and perforin [29, 30], suggesting the possibility of direct elimination through cytotoxicity of cells relevant to host protection from virus infection, or perhaps regulatory cells that are capable of restricting pathology. During LCMV infection, NK cells eliminate activated antigen-specific CD4+ T cells, which in turn dampens the CD8+ T-cell response to LCMV [13]. Alternatively, NK cells may indirectly affect lung pathology through the secretion of cytokines and/or chemokines and altering cell interactions and inflammatory responses. The production of IFN-γ by NK cells in lung may be relevant, as IFN-γ is known to limit CD8+ T-cell responses [37].

cruzi, an event that is not uncommon [1, 3] (Fig. 4A). Antibody titre to the three CH5424802 cost Trk receptors decreased sharply six months afterwards, when the patient was shifting to the chronic phase, and became undetectable 15-16 years after the start of the accidental infection (Fig. 4A), while the patient remains asymptomatic. The patient continues to be infected with T. cruzi and thus with Chagas’ infection because of the high antibody titres to the parasite >15 years after the onset of infection

(Fig. 4B). This illustrates that a Trk-Ab-seropositive patient in the acute phase can be converted to Trk-Ab-seronegative, consistent with 100% patients bearing acute Chagas’ disease Trk autoantibodies and with some patients (∼20%)

converting to Trk-Ab-seronegative when progressing to the chronic phase of the disease. In sum, our results show that individuals acutely infected with T. cruzi produce autoantibodies specific to TrkA, TrkB and TrkC, the tyrosine kinase receptors of the neurotrophins NGF, BDNF and NT-3 that regulate development and repair find more of central and peripheral nervous system [9, 16]. They were elicited by patients as young as a 4-year-old child and as aged as a 66 year-old adult and, thus, by an age-independent process. Given that acute infection starts after the parasite gains access to humans and lasts only a few months, the Trk autoantibodies should arise relatively soon after T. cruzi infection.

This was dramatically demonstrated in a patient with Chagas’ disease accidentally infected in a laboratory, as high titres of antibodies were evident less than two months after the accident (Fig. 4A). The Trk autoantibodies from patients with acute Chagas’ disease bear characteristics of antibodies produced in acute disease or after primary immunization (IgM and IgA isotype and low avidity). Unravelling the immunochemistry and biology of these neurotrophin receptor 5FU autoantibodies is of great interest because one of the autoantigens – TrkA – serves as a vehicle for T. cruzi, via its trans-sialidase/neurotrophic factor, to promote neuron survival [11] and to invade cells [10], while another autoantigen – TrkC – is used to induce survival and differentiation of neurons and Schwann cells [12]. ATA isolated from sera of patients in the indeterminate phase of Chagas’ disease compete with T. cruzi for Trk binding and inhibit infection in vitro and in vivo [13]. Consequently, ATA could modulate Chagas’ disease progression by reducing tissue parasitism in chronically infected individuals. In view of the present findings, ATA could play a role in the dramatic decline in tissue parasitism when patients progress from acute to chronic disease. This work was supported by NIH Grants NS40574 and NS42960.

The full-length cystatin Rapamycin cDNA obtained by RACE was subcloned into expression plasmid vector pET32a and expressed in Escherichia coli (Origami) as a protein fused

to a leader sequence of Tobacco Etch virus (TEV) protease and six histidines. The recombinant fusion protein was purified from E. coli lysate by affinity chromatography using chelating Sepharose FF resin (GE Healthcare, Uppsala, Sweden). The His-peptide in the fusion protein was cut off by TEV protease (kindly provided by Dr J. Liu, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China). The purity of the protein obtained was determined by SDS–PAGE and silver staining. The activities of cysteine proteases, cathepsin B, C, L and S, was measured following the Panobinostat methods as described by others with some modifications.[25] Bovine cathepsin B and C were purchased from Sigma and human cathepsin L and S were purchased from Calbiochem (Shanghai, China) and

Enzo (New York, NY), respectively. The fluorogenic substrates for cathepthin B (Z-Arg-Arg-AMC; Sigma–Aldrich), cathepsin C (Gly-PhE-naphthylamide; Sigma-Aldrich), cathepsin S (Z-Phe-Arg-7-amido-4- methylcoumarin; Calbiochem) and cathepsin L (Z-Phe-Arg-7-amido-4-methyl coumarin; Calbiochem) were obtained from individual suppliers. To measure the inhibition activity of rHp-CPI, the protease was incubated with substrate in the absence or presence of serially diluted rHp-CPI in appropriate buffer for 15 min. The amount of product was measured fluorometrically PAK5 with excitation at 360 nm and emission at 460 nm using a multiwall fluorescence spectrometer (Bio-Tek, Synergy HT, Corning, NY). Monoclonal antibody (mAb) against rHp-CPI was generated following the standard protocol.[26] Briefly, female BALB/c mice were immunized subcutaneously with 40 μg rHp-CPI emulsified in complete Freund’s adjuvant (Sigma-Aldrich) and boosted twice at 4-week interval with 20 μg rHp-CPI in incomplete Freund’s adjuvant. Spleen cells were isolated from the immunized

BALB/c mice 1 week after final boosting, and fused with logarithmically growing SP2/0 myeloma cells at a ratio of 1 : 1 in the presence of polyethylene glycol 1500 (Roche, Basle, Switzerland). The treated cells were re-suspended in RPMI-1640 medium supplemented with 20% fetal calf serum, OPI (oxaloacetate, pyruvate, insulin) and HAT (hypoxanthine, aminopterin, thymidine) media supplements (Sigma-Aldrich) and plated into 96-well tissue culture plates at a density of 2·0 × 105 cells per well in a volume of 200 μl. After culturing at 37° with 5·0% CO2 for 7–10 days, the culture wells were screened using indirect ELISA for the presence of anti-rHp-CPI antibody. The cells in positive wells were collected and subjected to cloning by limited dilution. The cloned hybridoma cells were injected into the peritoneal cavity of naive mice.