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.

Is it recurrent cryptococcal meningitis or immune reconstitution inflammatory syndrome?

We report a case of a 45-year-old patient with a history of cryptococcal meningitis who was started on antiretroviral therapy. The patient presented four months later with complaints of fever and memory loss. Lumbar puncture revealed positive cryptococcal antigen and therefore the patient was treated for recurrent cryptococcal meningitis. Unfortunately, the patient did not improve even after two weeks. The diagnosis of immune reconstitution was made at this time and steroids were started. The patient showed remarkable improvement.

Direct antiglobulin test reactive with complement only in warm type autoimmune hemolytic anemia.

Direct antiglobulin test (DAT) with only complement detected on red blood cells is a rare laboratory finding, and its significance in the setting of warm autoimmune hemolytic anemia (AIHA) is controversial. During 2 years (2003-2004) 277 patients with positive DAT were recorded in the blood bank registries, 17 of them had DAT reactive with C3 alone with no cold agglutinin or other nonimmune causes for hemolysis diagnosed. Red cell eluate disclosed small amounts of IgG in two patients. In nine patients no signs of clinical hemolysis were found, however, all these patients had underlying conditions that are known to be associated with red cells autoantibodies (autoimmune disorder or malignancy). Eight patients developed AIHA, seven of them with severe hemolysis. Three patients had idiopathic AIHA, and the others have been diagnosed with infectious, lymphoproliferative and autoimmune disorders. In two patients with acute infection the hemolytic process spontaneously resolved, three responded to corticosteroid therapy, while three patients were refractory to two lines of drug therapy and underwent splenectomy. Reticulocytopenia was found in four patients. Our results emphasize that AIHA with DAT reactive with complement alone is a rare disorder and might be accompanied by severe, refractory to conventional treatment and life-threatening hemolysis.

Pharmacological interventions for treating acute heterotopic ossification.

BACKGROUND: Heterotopic ossification (HO) is the formation of mature lamellar bone in soft tissue sites outside the skeleton. HO frequently complicates burns, arthroplasty, fractures, and spinal cord and brain injuries. It can impair joint function. OBJECTIVES: To determine the efficacy of medications to treat acute HO on radiological, symptomatic, functional impairment, and disability outcomes. SEARCH STRATEGY: We searched the Cochrane Musculoskeletal Injuries Group specialised register, the Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 2, 2004), MEDLINE (1966 to August 2004), EMBASE (1980 to August 2004), CINAHL (1982 to August 2004), other databases, reference lists of articles, and contacted trialists and drug companies. No language restrictions were applied. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials that assessed the efficacy of any medication for treating acute HO (confirmed by bone scintigraphy, radiography, ultrasonography, or biopsy) and which used radiography to grade post-treatment HO severity. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed the study quality and extracted data. We analysed two dichotomous outcomes: no progression in HO grade (versus progression) and improvement in HO grade (versus no improvement). MAIN RESULTS: Two randomised trials comparing disodium etidronate versus placebo were included (Ono 1988; Stover 1976), from which ninety-two participants with spinal cord injury had radiographically-proven HO at baseline. At the completion of the 12 week intervention, the Ono study but not the Stover study, suggested that disodium etidronate was associated with a significantly greater likelihood of successfully preventing the progression of radiographic HO grade, (relative risk (RR) 1.50; 95% confidence interval (CI) I 1.16 to 1.93; and RR 1.48; 95% CI 0.78 to 2.84 respectively) and a significantly greater likelihood of improvement in HO grade (RR 2.78; 95% CI 1.66 to 4.66; and RR 0.71; 95% CI 0.20 to 2.53 respectively). There was evidence of statistical heterogeneity for the latter outcome. Longer term radiographic, clinical or side effect outcomes were unavailable. Data was not pooled due to this heterogeneity and the inadequate duration of follow up. REVIEWERS' CONCLUSIONS: Given the absence of long term radiographic outcomes in the included studies, there is insufficient evidence to recommend the use of disodium etidronate or other pharmacological agents for the treatment of acute HO. It has been previously suggested that disodium etidronate acts by delaying, rather than preventing, the mineralization of HO, and that mineralization may occur after treatment cessation in many cases, thereby negating the benefit of disodium etidronate on eventual HO grade. Further studies are required to assess all pharmacological treatments for acute HO with sufficient follow-up duration.

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.

Successful treatment of aggressive HIV-associated non-Hodgkin's lymphoma with combination chemotherapy, biotherapy with rituximab and HAART: presentation of a therapeutic option.

The incidence of non-Hodgkin's lymphoma (NHL) in individuals infected with human immunodeficiency virus (HIV) is more than 60 times higher than in matched controls. In the vast majority of cases aggressive pathological subtypes and advanced stages prevail, extranodal sites are involved and systemic symptoms are present. The prognosis of HIV-NHL remains poor and the optimal therapeutic approach has yet to be defined. We report a 48-year-old Ethiopian woman with advanced-stage HIV infection, who developed diffuse large cell, immunoblastic type B-cell NHL and was treated with a modified CHOP-like chemotherapy combined with Rituximab and supported with growth factor. Highly active antiretroviral therapy (HAART) and opportunistic infections prophylaxis were administered concomitantly. The patient completed 6 cycles of therapy and currently, 76 weeks after diagnosis, is in complete clinical remission. Despite the fact that there was a transient decrease in the CD4-positive cell number and a 1.5 log increase in plasma viral load there were no opportunistic infections, nor was life-threatening toxicity seen. Rituximab seems a well-tolerable and advantageous adjunct to chemotherapy and HAART in the treatment of aggressive HIV-associated NHL andshould be investigated in large trials in the future.

Evidence that inhibition of cathepsin-B contributes to the neuroprotective properties of caspase inhibitor Tyr-Val-Ala-Asp-chloromethyl ketone.

During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certain caspase inhibitors. Here we report our studies to identify a target for and hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-alanine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neuronal cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from cell death in response to oxidative stress and oxygen/glucose deprivation. Affinity labeling with biotinylated YVAD-cmk demonstrated distinct binding proteins for the inhibitor in cells from the central nervous system versus Jurkat cells. Binding to the novel target protein was displaced by class-specific protease inhibitors and suggested that the target is a cysteine protease. Affinity purification and sequencing identified the target as cathepsin-B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the target protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-B inhibitors rescue hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.

[Effective treatment with Lamivudine of patients with reactivation of hepatitis B following chemotherapy administration].

Chemotherapy administration to patients with lymphoproliferative diseases that are carriers of hepatitis B can be complicated by reactivation of Hepatitis B. This may lead to morbidity and mortality due to liver failure. We report 2 cases, treated recently. The first case is that of a 63-year-old female with a diagnosis of immunoblastic lymphoma. The patient was treated with the ProMACE-CytaBOM protocol. During treatment Hepatitis B was reactivated and after termination, of chemotherapy she developed fulminant hepatitis with hyperbilirubinemia, coagulopathy, hypoalbuminemia and ascites. The second case is that of a 34 years old male with a diagnosis of T-ALL who was treated according to the BFM 95 protocol. He had reactivation of Hepatitis B during induction therapy. These two patients were treated with Lamivudine with resolution of the hepatitis and disappearance of HBV DNA from the sera. Prophylactic administration of Lamivudine enabled reinduction of chemotherapy in the first case after relapse of the lymphoma and continuation of BFM 95 protocol in the second patient. Lamivudine inhibits replication of hepatitis B virus and prevents reactivation of Hepatitis B during immunosuppression induced by chemotherapy and probably ameliorates the severity of already reactivated hepatitis.

A case of transvaginal evisceration.

We present a case of spontaneous evisceration of the small bowel through the vaginal vault in a 61-year-old women. The predisposing factors and management are discussed.

Bryostatin 1-tamoxifen combinations show synergistic effects on the inhibition of growth of P388 cells in vitro.

This study shows that combinations of bryostatin 1, a novel modulator of protein kinase C currently under clinical evaluation, with the anti-oestrogenic agent tamoxifen caused a large synergistic enhancement of growth inhibition in P388 cells in vitro. The growth-inhibitory effects of bryostatin 1 in the presence of non-inhibitory concentrations of tamoxifen were increased by approximately 200-fold, whereas growth inhibition by tamoxifen in the presence of non-inhibitory concentrations of bryostatin 1 were increased over 30-fold. These data have been confirmed by isobologram analysis. The precise mechanism underlying this effect is unknown, although preliminary data implicating protein kinase C is presented. The magnitude of this synergistic effect, together with evidence of clinical responses seen when these agents were given sequentially in ovarian cancer, merits further study.

Abnormal cervical cytology leading to the diagnosis of a primary serous adenocarcinoma of the peritoneum.

A case is presented where abnormal cervical cytology in an asymptomatic female led to the diagnosis of a primary serous adenocarcinoma of the peritoneum. We would like to highlight the importance to the physician of a tumour diathesis on malignant cervical cytology.

Comparison of PCR/nucleic acid hybridization and EIA for the detection of Chlamydia trachomatis in different populations in a regional centre.

Culture on McCoy cell monolayers has been accepted as the reference method for the detection of Chlamydia trachomatis. Recent studies have shown that polymerase chain reaction (PCR)/nucleic acid hybridization based methods have increased sensitivity over culture while still retaining specificity. In situations where organism viability is of concern, due to factors such as transportation delays, culture is inappropriate. Regional laboratories therefore have not been able to utilize the reference method and have been forced to use less reliable methods. The aims of our study were to assess the feasibility of performing PCR to diagnose infections due to C. trachomatis in a regional laboratory using a new commercial kit--Amplicor (Roche Molecular Systems, Branchburg, NJ) and to compare the current enzyme-immunoassay (EIA) based-methods used in our laboratory (VIDAS [bioMerieux Vitek, Hazelwood MO] and IDEIA [Novo Nordisk Diagnostics, Cambridge, UK]) against PCR. Thirteen positive Amplicor specimens were found in 267 urine specimens collected from asymptomatic adolescent males and females. All 13 were confirmed positive using major outer membrane protein gene PCR (MOMP). VIDAS and IDEIA showed 100% correlation to each other but only detected 5/13 positives. Of 140 consecutive patients attending the regional sexual health clinic, 13 were Amplicor positive, 11/13 MOMP positive and 10/13 positive by VIDAS. Five of 254 patients attending the hospital antenatal clinic were positive by Amplicor, all being confirmed by MOMP. No PCR inhibition was detected in a random sample of 100 varied negative Amplicor tests using a modification of the Amplicor kit. No contamination was experienced. The Amplicor kit was shown to be suitable for use in the routine clinical laboratory with minimal disruption to workflow. For regional laboratories this kit should provide more accurate results than EIA based methods, particularly in the detection of asymptomatic persons.

Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1.

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Cross-resistance studies on two K562 sublines resistant to diaziridinylbenzoquinones.

Two resistant K562 sublines have been developed by treatment with AZQ (2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone) and BZQ (2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone). The ID50 values of for AZQ on K562, the AZQ-resistant sublines (AZQR) and the BZQ-resistant sublines (BZQR) were 0.063, 1.47 and 0.244 microM, respectively. The relative ID50 values for BZQ on the same cell lines were 0.2, 0.67 and 0.83 microM, respectively. Although it is generally believed that these two quinones function by different mechanisms, the two sublines have similar decreased levels of cytochrome P-450 reductase and DT-diaphorase and increased levels of glutathione and superoxide dismutase, compared to the parent cell line. The sublines are also cross-resistant to adriamycin, mitozolamide, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and mitomycin C. This work indicates the potential multifactorial mechanisms by which drug resistance can be induced in cell lines in the absence of conventional 'P'-glycoprotein multidrug resistance.

The effect of phytic acid on the resolution of peptides and proteins in capillary electrophoresis.

Studies are reported on the effect of the sodium salt of phytic acid on the resolution of peptides and proteins. Improved separation in the case of peptides is shown to be due to ion-ion pairing interactions between the positively charged peptides and the phytic acid polyanionic species. The improved peak shapes related to the proteins can be interpreted in terms of the sample preconcentration due to injection of analytes from a water medium to one of high ionic strength.