The PTPROt tyrosine phosphatase functions as an obligate haploinsufficient tumor suppressor in vivo in B-cell chronic lymphocytic leukemia.

The tyrosine phosphatase PTPROt is a suggested tumor suppressor (TS) in B-cell chronic lymphocytic leukemia (CLL), and its expression is reduced in this disease. In order to examine how reduced PTPROt expression affects CLL in vivo we induced CLL in PTPROt-targeted mice. Unexpectedly, loss of both Ptprot alleles delayed disease detection and progression and lengthened survival relative to mice carrying two intact alleles, indicating that PTPROt fulfills a novel tumor-promoting role in CLL. Tumor cells from mice lacking PTPROt exhibited reduced B-cell receptor (BCR)-induced signaling, as well as increased apoptosis and autophagy. Inhibition of BCR/Src signaling in CLL cells induced their apoptosis, indicating that these findings are linked causally. These results suggest a cell-autonomous mechanism for the weakened CLL phenotype of PTPROt-deficient mice and uncover non-redundant roles for PTPROt in support of BCR signaling and survival of CLL cells. In contrast, loss of only one Ptprot allele induced earlier detection and progression of CLL and reduced survival, consistent with a tumor-suppressing role for PTPROt. Tumor cells from mice lacking one or both Ptprot allele exhibited increased interleukin-10 (IL-10) expression and signaling, factors known to support CLL; cells lacking one Ptprot alleles exhibited normal BCR signaling and cell death rates. We conclude that loss of one Ptprot allele promotes CLL, most likely by activating IL-10 signaling. Loss of both Ptprot alleles also reduces BCR signaling and increases cell death rates, offsetting the IL-10 effects and reducing the severity of the disease. PTPROt thus functions as an obligate haploinsufficient TS in CLL, where its expression levels determine its role as a promoter or inhibitor of the tumorigenic process in mice. Partial loss of PTPROt generates the strongest disease phenotype, suggesting that its intermediate expression levels in CLL are selected for.

CD84 mediates CLL-microenvironment interactions.

Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

CD84 is a survival receptor for CLL cells.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

Can parametrectomy be avoided in early cervical cancer? An algorithm for the identification of patients at low risk for parametrial involvement.

AIMS: To assess the rate of parametrial involvement in a large cohort of patients who underwent radical hysterectomy for cervical cancer and to suggest an algorithm for the triage of patients to simple hysterectomy or simple trachelectomy. METHODS: Multicenter retrospective study of patients with cervical cancer stage I through IIA who underwent radical hysterectomy and pelvic lymphadenectomy. The patients were divided into 2 groups according to whether or not the parametrium was involved. The two groups were compared with regard to the clinical and histopathological variables. Logistic regression of the variables potentially assessable prior to definitive hysterectomy such as age, tumor size, lymph-vascular space invasion (LVSI) and nodal involvement was performed. RESULTS: Five hundred and thirty patients had specific histological data on parametrial involvement and in 58 (10.9%) patients, parametria was involved. Parametrial involvement was significantly associated with older age, tumors larger than 2 cm, deeper invasion, LVSI, involved surgical margins, and the presence of nodal metastasis. By triaging patients with a tumor ≤ 2 cm and no LVSI, the parametrial involvement rate was 1.8% (2/112 patients). With further triage of patients with negative nodes, the rate of parametrial involvement was 0% (0/107 patients). CONCLUSION: Using a pre-operative triage algorithm, patients with early small lesions, no LVSI and no nodal involvement may be spared radical surgical procedures and parametrectomy. Further prospective data are urgently needed.

CCL2 (pM levels) as a therapeutic agent in Inflammatory Bowel Disease models in mice.

BACKGROUND: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation-induced colorectal cancer. METHODS: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane-DSS (AOM-DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. RESULTS: Daily administration of CCL2 (60-120 ng) significantly decreased the development of TNBS- and DSS-induced colitis. In a DSS-AOM model, CCL2-treated mice developed significantly fewer tumors (P < 0.005) at 11 weeks. Chronic inflammation in the CCL2-treated mice was significantly less pronounced as compared to phosphate-buffered saline-treated mice. CONCLUSIONS: Administration of pM levels of CCL2 significantly inhibits migration of T cells in amelioration of TNBS and DSS colitis and inhibits development of colorectal cancer in an AOM-DSS colitis model in mice. Thus, pM levels of CCL2 may be clinically beneficial as an antiinflammatory agent in IBD.

A multicenter validation of computerized tomography models as predictors of non- optimal primary cytoreduction of advanced epithelial ovarian cancer.

AIMS: To compare the validity of four predictive models of preoperative computerized tomography (CT) scans in predicting suboptimal primary cytoreduction in patients treated for advanced ovarian cancer. PATIENTS AND METHODS: Preoperative CT scans of patients with stage III/IV epithelial ovarian cancer who underwent primary cytoreductive surgery at one of four medical centers were reviewed by radiologists blinded to surgical outcome. The validity of each set of CT criteria previously published by Nelson, Bristow, Dowdy, and Qayyum as predictors of suboptimal cytoreduction was assessed. RESULTS: Data of 123 patients were evaluated. Optimal cytoreduction (largest diameter of residual tumor < or =1cm) was obtained in 90 (73.2%) patients. All CT models were able to significantly predict surgical outcome (p<0.02). The respective sensitivity, specificity, and accuracy of the CT models to predict sub-optimal cytoreduction was 64%, 64% and 64% for Nelson's criteria, 70%, 64% and 66% for Bristow's criteria, 79%, 60%, and 65% for Dowdy's criteria, and 67% 57% and 60% for Qayyum's criteria. CONCLUSIONS: Apart from Dowdy's criteria, the accuracy rates of CT predictors of suboptimal cytoreduction in the original cohorts could not be confirmed in this cross validation. This study underscores the difficulty in devising universally applicable selection criteria or models that reliably predict surgical outcome across institutions and surgeons.

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

Grb7 expression and cellular migration in chronic lymphocytic leukemia: a comparative study of early and advanced stage disease.

Grb7, a noncatalytic intracellular adaptor protein involved in cell migration, is overexpressed in certain invasive and metastatic solid tumors. We found a highly significant difference in the level of expression of Grb7 between chronic lymphocytic leukemia (CLL) cells obtained from stage I and stage IV patients (P<0.001). Using semiquantitative RT-PCR, we detected high levels of Grb7 in 88% of stage IV patients vs only 18% in stage I patients. A corresponding increase was found in the in vitro migration of stage IV CLL cells in comparison to stage I cells. The statistically significant difference in the expression of Grb7 between stage IV and stage I patients was preserved even when tested specifically in the ZAP70-positive group (P<0.01). These findings show that Grb7 levels reflect the severity of the disease, and may be used, in conjunction with ZAP70, to predict disease progression.

Low levels of IFN-gamma down-regulate the integrin-dependent adhesion of B cells by activating a pathway that interferes with cytoskeleton rearrangement.

In order to fully mature and participate in the humoral immune response, immature B cells must first migrate into specific areas in the spleen where they differentiate into mature cells. However, before their maturation in the spleen, immature B cells must be excluded from non-splenic secondary lymphoid organs where any antigen encounter would lead to the death of the cells because of the negative selection process. We have recently shown that immature B cells can actively exclude themselves from antigen-enriched sites by down-regulating their integrin-mediated adhesion in a process mediated by interferon-gamma (IFN-gamma). In this study, we followed the pathway by which IFN-gamma regulates the homing of B cells. We show here that the inhibitory signal of IFN-gamma is transmitted through the IFN-gamma receptor whose engagement leads to the activation of PI3K. This PI3K activation subsequently leads to the inhibition of PKCalpha phosphorylation and cytoskeleton rearrangement required for promoting integrin-mediated adhesion and migration of B cells.

Huge acoustic neurinomas presenting in the late stage of pregnancy. Treatment options and review of literature.

BACKGROUND: Even though vestibular schwannomas rarely present during pregnancy, symptoms may appear or worsen particularly in this period. The clinical picture may include tinnitus, hearing abnormalities, and in large tumors, brain-stem and cerebellar compression with involvement of additional cranial nerves. Large vestibular schwannomas (also known as Acoustic Neurinomas) present a great challenge in peripartum management of both the mother and the fetus. MATERIAL AND METHOD: We present a case of a 24-year old woman, with headache, papilledema, ataxia, and multiple cranial nerve weakness, diagnosed in the 35th week of pregnancy. MRI demonstrated a huge vestibular schwannoma compressing the brainstem and causing obstructive hydrocephalus. RESULT: In the presence of high intra-cranial pressure a ventriculo-peritoneal shunt was first inserted, enabling delay of tumor surgery until after delivery. A successful elective cesarean section followed at 37 weeks, and radical tumor surgery was performed a week later. Maternal and fetal outcome were excellent. DISCUSSION: The options, sequence and timing of the neurosurgical and obstetrical interventions are discussed. Other reports of large vestibular schwannomas that presented during pregnancy are reviewed. Advances in neurosurgery, neuroradiology, neuroanesthesiology and obstetrics are highlighted, and their impact on outcome is discussed in comparison to the poor results reported in the past. Emphasis is made on the importance of early diagnosis, that necessitates high-index of suspicion by the obstetrician, in any pregnant woman presenting abnormal neurological signs. CONCLUSION: We conclude that with a cooperative team approach, maternal and fetal prognosis can today be excellent, even in cases of large vestibular schwannomas diagnosed in the late stage of pregnancy.

Invariant chain controls H2-M proteolysis in mouse splenocytes and dendritic cells.

The association of invariant (Ii) chain with major histocompatibility complex (MHC) class II dimers is required for proper antigen presentation to T cells by antigen-presenting cells. Mice lacking Ii chain have severe abnormalities in class II transport, T cell selection, and B cell maturation. We demonstrate here that H2-M, which is required for efficient class II antigenic peptide loading, is unexpectedly downregulated in splenocytes and mature dendritic cells (DCs) from Ii(-/-) mice. Downregulation reflects an increased rate of degradation in Ii(-/-) cells. Degradation apparently occurs within lysosomes, as it is prevented by cysteine protease inhibitors such as E64, but not by the proteasome inhibitor lactacystin. Thus, Ii chain may act as a lysosomal protease inhibitor in B cells and DCs, with its deletion contributing indirectly to the loss of H2-M.

Degradation of distinct assembly forms of immunoglobulin M occurs in multiple sites in permeabilized B cells.

Protein degradation is essential for quality control which retains and eliminates abnormal, unfolded, or partially assembled subunits of oligomeric proteins. The localization of this nonlysosomal pre-Golgi degradation to the endoplasmic reticulum (ER) has been mostly deduced from kinetic studies and carbohydrate analyses, while direct evidence for degradation within the ER has been provided by in vitro reconstitution of this process. In this article, we took advantage of the transport incompetence of permeabilized cells to directly demonstrate that the selective degradation of secretory IgM (sIgM) in B lymphocytes is transport-dependent. We show that, upon permeabilization of the plasma membrane with either streptolysin O or digitonin, sIgM is not degraded unless transport is allowed. Nevertheless, upon complete reduction of interchain disulfide bonds with thiols, the free mu heavy chains are degraded by a transport-independent quality control mechanism within the ER. This latter degradation is nonselective to the secretory heavy chain mus, and the membrane heavy chain mum, which is normally displayed on the surface of the B cell, is also eliminated. Moreover, the degradation of free mus is no longer restricted to B lymphocytes, and it takes place also in the ER of plasma cells which normally secrete polymers of sIgM. Conversely, when assembled with the light chain, the degradation is selective to sIgM, is restricted to B lymphocytes, and is a transport-dependent post-ER event.

Requirement for invariant chain in B cell maturation and function.

Previously the role of invariant chain (Ii) had been described only as a chaperone that facilitates folding and transport of major histocompatability complex class II molecules; here it is shown that Ii is required for B cell development. B cells from mice lacking Ii were found to have a low response to T-independent type II antigen and could not proliferate after the mice were injected with antigen. Study of cell surface markers revealed a developmental arrest that prevented immature virgin B cells from becoming mature B cells in the periphery. This block was independent of major histocompatability complex class II expression and was an intrinsic feature of B cells that correlated with the amount of Ii. Thus, Ii participates by an unknown mechanism in B cell maturation.

Ruptured tubo-ovarian abscess complicating transcervical cryopreserved embryo transfer.

OBJECTIVE: To present a rare infectious complication related to transcervical ET, without prior transvaginal puncture. DESIGN: Case report. SETTING: Hadassah University Hospital, IVF-ET unit. PATIENT: One patient undergoing cryopreserved-thawed ET. INTERVENTIONS: Artificial preparation of the endometrium with E2 and P, followed by transcervical intrauterine cryopreserved-thawed embryo transfer. RESULTS: After ET, severe pelvic inflammatory disease (PID) with ruptured tubo-ovarian abscess was diagnosed and treated. CONCLUSIONS: Severe PID including tubo-ovarian abscess formation should be considered a potential complication after ET, even without transvaginal oocyte aspiration.

Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

Treatment of vulvar lichen sclerosus in the elderly: an update.

Lichen sclerosus et atrophicus is a disorder of the skin that can occur anywhere on the body and in all age groups but mainly affects middle-aged and elderly women in the vulvoperineal area. It consists of ivory or pink papules or macules that eventually coalesce into thin, gray, parchment-like areas. Clinically, the main symptoms are severe and intractable itching and vaginal soreness with dyspareunia. Although it has been described to be associated with an increased risk for epithelial malignancy this, in fact, very rarely occurs. The exact nature of LSA is still unknown. The accumulation of evidence does little to clarify its pathogenesis and etiology. The different reports indicate at least three general possibilities; autoimmune, metabolic, and more recently infectious etiology. The coexistence of such diverse findings in one disease entity may indicate one of the two; either we are facing a group of very similar conditions, which will be separated in the future into several closely related clinical entities, each with its own etiology, or that all findings represent a complex multi-step single pathogenetic mechanism. The latter possibility seems more probable because it has previously been suggested that B. burgdorferi, a recent prime suspect in the pathogenesis of LSA, may induce both metabolic and autoimmune abnormalities in the course of infection. New therapeutic options and attitudes emerge that dramatically improved the conservative treatment of this disease (Table 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.

Post-translational regulation of IgM expression in B lymphocytes. Selective nonlysosomal degradation of assembled secretory IgM is temperature-dependent and occurs prior to the trans-Golgi.

B cells express on their surface the membrane form of IgM (mIgM). Upon differentiation, the resulting plasma cells synthesize and secrete large amounts of the secretory form of IgM (sIgM). Surprisingly, B lymphocytes synthesize an excess of secretory mu chain over the expressed membrane mu chain. However, the sIgM is degraded intracellularly, indicating regulation of IgM expression at the post-translational level. In the present report, we show that the assembly, maturation, and degradation of IgM in 38C B lymphocytes are highly accelerated above a certain threshold temperature. Furthermore, the degradation of sIgM is delayed and takes place by the time the maturation of mIgM in the trans-Golgi is almost completed. Neither chloroquine nor monensin has any effect on this degradation, demonstrating a nonlysosomal pre-trans-Golgi process. In addition, the degradation is of endoglycosidase H-sensitive assembled sIgM molecules. We conclude that the degradation of sIgM in 38C B lymphocytes is a postendoplasmic reticulum, pre-trans-Golgi process. We suggest that this degradation process plays a role in the post-translational regulation of expression of soluble lumenal sIgM.