Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A.

INTRODUCTION: The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. AIM: To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. METHOD: HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg(-1) rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. RESULTS: Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. CONCLUSION: FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before.

Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A.

BACKGROUND: Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. OBJECTIVES: Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. METHODS: Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. RESULTS: In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. CONCLUSIONS: Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response.

Human Atopic Dermatitis Skin-derived T Cells can Induce a Reaction in Mouse Keratinocytes in vivo.

In atopic dermatitis (AD), the inflammatory response between skin-infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice through keratinocyte activation and consequently cause the development of eczematous lesions. Punch biopsies of the lesional skin from AD patients were used to establish skin-derived T cell cultures, which were transferred to NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that the subcutaneous injection of the human AD skin-derived T cells resulted in the migration of the human T cells from subcutis to the papillary dermis followed by the development of erythema and oedema in the mouse skin. Furthermore, the human T cells induced a transient proliferative response in the mouse keratinocytes shown as increased numbers of Ki-67(+) keratinocytes and increased epidermal thickness. Out of six established AD skin-derived T cell cultures, two were superior at inducing a skin reaction in the mice, and these cultures were found to contain >10% CCR10(+) T cells compared to <2% for the other cultures. In comparison, blood-derived in vitro-differentiated Th2 cells only induced a weak response in a few of the mice. Thus, we conclude that human AD skin-derived T cells can induce a reaction in the mouse skin through the induction of a proliferative response in the mouse keratinocytes.

Calibration of bioelectrical impedance analysis for body composition assessment in Ethiopian infants using air-displacement plethysmography.

BACKGROUND/OBJECTIVES: Assessment of infant body composition (BC) is crucial to understand the consequences of suboptimal nutritional status and postnatal growth, and the effects of public health interventions. Bioelectrical impedance analysis (BIA) is a feasible, relatively inexpensive and noninvasive method for assessing BC. However, very little research has been conducted in low- and middle-income populations, where efforts to prevent or treat malnutrition in early life are a public health priority. We aimed to develop equations for predicting fat-free mass (FFM) and fat mass (FM) based on BIA in 0- to 6-month-old Ethiopian infants. SUBJECTS/METHODS: The study comprised a total of 186 BC assessments performed in 101 healthy infants, delivered at Jimma University Specialized Hospital. Infant air-displacement plethysmography (IADP) was the criterion method, whereas weight, length, sex, age and an impedance index (L(2)/Z50) were predictors. Prediction equations were developed using stepwise multiple linear regression and the accuracy was evaluated with a 10-fold cross-validation approach. RESULTS: A linear regression model based on body weight, age and sex predicted FFM, estimated by IADP, with an adjusted R(2) and root mean square error (RMSE) of 0.94 and 200 g, respectively. Adding impedance index to the model resulted in a significantly improved model fit (R(2)=0.95; RMSE=181 g). For infants below 3 months of age, inclusion of impedance index did not contribute to an improved model fit for predicting FFM compared with a model already comprising weight, sex and age. CONCLUSIONS: The derived equations predicted FFM with acceptable accuracy and may be used in future field surveys, epidemiological studies and clinical trials conducted in similar sub-Saharan African population groups aged 0-6 months.

Differential serodiagnostics of Toxocara canis and Toxocara cati--is it possible?

One of the most common zoonotic helminth infections is caused by species in the genus Toxocara, particularly Toxocara canis and T. cati (Syn. T. mystax). However, their relative contribution to toxocarosis in humans remains largely unknown because causative larvae are seldom recovered and uncertainties regarding the validity of existing serological assays. In this study, we used sera from a pig model experimentally infected with T. canis and T. cati to evaluate whether a Western blot could discriminate between the two species. No proteins were observed that could be used as a diagnostic tool. In addition, a heterogenic protein pattern between individual hosts was found, which was most pronounced in the T. cati-infected pigs. There is therefore an urgent need to optimize and validate current methods or develop new species-specific serological methods in order to implement appropriate control measures.

Depletion of regulatory T cells in a hapten-induced inflammation model results in prolonged and increased inflammation driven by T cells.

Regulatory T cells (Tregs ) are known to play an immunosuppressive role in the response of contact hypersensitivity (CHS), but neither the dynamics of Tregs during the CHS response nor the exaggerated inflammatory response after depletion of Tregs has been characterized in detail. In this study we show that the number of Tregs in the challenged tissue peak at the same time as the ear-swelling reaches its maximum on day 1 after challenge, whereas the number of Tregs in the draining lymph nodes peaks at day 2. As expected, depletion of Tregs by injection of a monoclonal antibody to CD25 prior to sensitization led to a prolonged and sustained inflammatory response which was dependent upon CD8 T cells, and co-stimulatory blockade with cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) suppressed the exaggerated inflammation. In contrast, blockade of the interleukin (IL)-10-receptor (IL-10R) did not further increase the exaggerated inflammatory response in the Treg -depleted mice. In the absence of Tregs , the response changed from a mainly acute reaction with heavy infiltration of neutrophils to a sustained response with more chronic characteristics (fewer neutrophils and dominated by macrophages). Furthermore, depletion of Tregs enhanced the release of cytokines and chemokines locally in the inflamed ear and augmented serum levels of the systemic inflammatory mediators serum amyloid (SAP) and haptoglobin early in the response.

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition.

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady-state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ(+) and CD8(+) T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild-type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ(+) T-cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.

Local and systemic effects of co-stimulatory blockade using cytotoxic T lymphocyte antigen-4-immunoglobulin in dinitrofluorobenzene- and oxazolone-induced contact hypersensitivity in mice.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA-4-Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA-4-Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA-4-Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose-dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)- and oxazolone-induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA-4-Ig. It was further shown that CTLA-4-Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA-4-Ig reduces infiltration of activated CD8(+) T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute-phase proteins in circulation. Finally, our results suggest that CTLA-4-Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA-4-Ig induces long-term immunosuppression of both DNFB- and oxazolone-induced inflammation and our data are the first to compare the effect of this compound in both DNFB- and oxazolone-induced CHS and to show that CTLA-4-Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.

Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand.

The molecular chaperone calreticulin has been shown to bind C1q and mannan-binding lectin (MBL), which are constituents of the innate immune defence system. C1q and MBL do not share a large sequence identity but have a similar overall molecular architecture: an N-terminal triple-helical collagen-like domain and a C-terminal globular domain with ligand-binding properties. C1q is a hetero-trimer, while MBL is a homo-trimer, but due to the presence of N-terminal cysteines they both form higher order oligomers of trimers, which are the mature functional molecules. The same molecular architecture is utilized by many other functionally diverse molecules and in this work the interaction of calreticulin with C1q and structurally similar molecules was investigated. In addition to C1q and MBL, CD40 ligand (CD40L), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) were found to bind calreticulin strongly. A low level or no binding was observed for adiponectin, tumour necrosis factor-alpha (TNF-alpha), CD30L, surfactant protein-A and -D and collagen VIII. The interaction with calreticulin required a conformational change in CD40L, TRAIL and FasL and showed the same characteristics as calreticulin's interaction with C1q and MBL: a time-dependent saturable binding to immobilized protein, which was initially sensitive to salt but gradually developed into a salt-insensitive interaction. Thus, the interaction requires a structural change in the interaction partner and leads to a conformational change in calreticulin itself. The implications of these results are that calreticulin may function as a general response modifier for a whole group of immunologically important proteins.

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.

A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

A comparison of first-void urine, self-administered low vaginal swab, self-inserted tampon, and endocervical swab using PCR tests for the detection of infection with Chlamydia trachomatis.

BACKGROUND: Polymerase chain reaction (PCR) tests, AMPLICOR, Roche Diagnostics, were shown to be an acceptable and sensitive method of detecting Chlamydia trachomatis infection. The PCR test's ability to evaluate different specimen types is worth determining, as well as the acceptability to Thai women of the self-collection of samples. METHODS: Of the 1011 subjects interviewed, 953/1011 subjects (94.3%) agreed to self-test, 523 were commercial sex workers (CSWs) and 430 were outpatient women (OPW). More than half [570/953 (59.8%)] participated in the four-specimen collection, to be tested by PCR for C. trachomatis. Specimens were collected via first-void urine (FVU), self-administered low vaginal swab (LVS), self-inserted tampon, and endocervical swab (ES). The majority, 906/953 subjects (95.1%), had only three methods of specimen collection, LVS being excluded. RESULTS: The prevalence of positive C. trachomatis detection among the CSWs/OPW was 17.6/7.2%, 15.6/5.4%, 12.8/4.2%, and 11.6/5.7% using tampons, LVS, FUV, and ES collection methods respectively. Tampons were used to compare results from other specimen types in both groups. Significantly more OPWs were willing to use a tampon for repeat specimen collection (85.1%) than were the CSWs (62.3%). Willingness to use a LVS again was not significant, 75.2% in outpatient women and 74% in CSWs. CONCLUSIONS: Tampon and LVS, self-collection methods are acceptable to women in Thailand and are a good alternative method for detection of C. trachomatis.

Prevalence of sexually transmitted infections among clients of female commercial sex workers in Thailand.

BACKGROUND: Clients of commercial sex workers are considered at high risk for the acquisition and transmission of sexually transmitted diseases (STDs). Identification and treatment of infections in this group could help to reduce the transmission of STDs. GOAL: To ascertain the prevalence of sexually transmitted organisms in male clients of female sex workers in Thailand by analysis of seminal fluid collected after intercourse. STUDY DESIGN: Used condoms were collected from 291 male clients attending a brothel in Hat Yai, Thailand during a 7-day period. Nucleic acid was extracted from seminal fluid and tested by polymerase chain reaction for the presence of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, herpes simplex virus (HSV) and HIV sequences. RESULTS: Overall, 17 (6%), 47 (16%), and 2 (1%) of specimens were positive for C trachomatis, N gonorrhoeae and T vaginalis respectively. HSV sequences were found in 24 (8%) of the specimens: 14 specimens (5%) with HSV type 1, and 11 specimens (4%) with HSV type 2. HIV RNA was detected in two samples (1%). Overall, 75 specimens (26%) were positive for one or more infections, and more than one pathogen was detected in 16 specimens (5%). CONCLUSION: This study reports a high rate of STDs among clients of female sex workers in Thailand. Consequently, this population is a significant risk for transmitting STDs to commercial sex workers and to other noncommercial partners. Strategies that target this population of men are needed to reduce STD and HIV transmission.

The prevalence of urethral infections amongst asymptomatic young men in Hat Yai, southern Thailand.

The aim of this study was to survey sexual behaviour and estimate the prevalence of urethral infections amongst male vocational college students. A cross-sectional survey was performed among 479 young men attending 2 vocational colleges in Hat Yai, southern Thailand. Polymerase chain reaction (PCR) tests of first-void urine (FVU) samples were used to detect infection with Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma genitalium and Mycoplasma hominis. Girlfriends were the usual sexual partners for 89% of men with only 11% regularly patronizing sex workers. Condom usage was low. The prevalence of any urethral infection was 15.9% with: C. trachomatis 4%, N. gonorrhoeae 0.2%, U. urealyticum 10.9%, M. genitalium 2.3% and M. hominis 1.3%. Infection with more than one organism was found in 2% of men. While the prevalence of infection with chlamydia or gonorrhoea was relatively low, the prevalence of 'any urethral infection' was moderately high and suggests that unprotected sexual intercourse is commonly occurring. As girlfriends were the most usual sexual partners, they must be at significant risk of pelvic infection. There is a need for programmes targeting this group of people.

Can a two-glass urine test or leucocyte esterase test of first-void urine improve syndromic management of male urethritis in southern Thailand?

The goal of this study was to determine whether a urine two-glass test or a leucocyte esterase (LE) test of first-void urine (FVU) improve the sensitivity or specificity of the World Health Organization (WHO) algorithm for the syndromic management of men with urethritis in southern Thailand. A secondary aim was to determine whether infection with Trichomonas vaginalis was sufficiently common to include treatment for it in a syndromic management protocol. One hundred and twenty-nine men with symptoms of urethritis seen at 2 STD clinics in Songkla Province, Thailand were enrolled. Symptoms and signs of each man were recorded and a urethral swab collected for microscopy and culture for Neisseria gonorrhoeae. A two-glass urine test and an LE test of an FVU specimen were performed. The FVU was tested by polymerase chain reaction (PCR) for N. gonorrhoeae, Chlamydia trachomatis and T. vaginalis. Dysuria was a symptom in 78% of men. A urethral discharge was a symptom in 68% but was evident on examination in 95% of the men. The prevalences of infection were 32.6% for N. gonorrhoeae, 23.3% for C. trachomatis, 1.6% for T. vaginalis and 51.9% for any infection. The sensitivities and specificities of urethral discharge on examination, two-glass test and LE test of FVU as indicators of infection with either or both of N. gonorrhoeae or C. trachomatis were 97% and 8%; 57% and 83%; and 59% and 78% respectively. Combinations of urethral discharge on examination and one of the other indicators were more specific but much less sensitive than the presence of discharge alone. Culture for N. gonorrhoeae was found to be only 43% sensitive compared with an expanded gold standard involving a PCR test. Our analysis demonstrates that neither the two-glass test nor the LE test of FVU were useful in improving on the WHO algorithm for management of men with urethritis. T. vaginalis was not common enough to include in a first-line syndromic management protocol for male urethritis. We recommend that, in southern Thailand, men with symptoms of urethritis in whom a urethral discharge is present on examination be offered immediate treatment for both N. gonorrhoeae and C. trachomatis as per the WHO algorithm.

IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells.

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.

Signal transduction by the major histocompatibility complex class I molecule.

Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell depending on the fine specificity of the anti-MHC-I antibodies, the context of CD8 ligation, the nature and cell cycle state of the MHC-I-expressing cell and the presence or absence of additional cellular or humoral stimulation. This paper reviews the biochemical, physiological and cellular events immediately after and at later intervals following MHC-I ligation. It is hypothesized that MHC-I expression, both ontogenically and in evolution, is driven by a cell-mediated selection pressure advantageous to the MHC-I-expressing cell. Accordingly, in addition to their role in T-cell selection and functioning, MHC-I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems.

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases.

In addition to providing the framework for peptide presentation, major histocompatibility complex class I (MHC-I) molecules can act as signal transducing molecules in lymphoid cells. Here we show that the mobilization of intracellular calcium, which follows crosslinking of MHC-I molecules on human B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine/threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells.