Development Of Opportunistic Infections Research Articles

Simian Immunodeficiency Virus Infection Potently Modulates Chemokine Networks and Immune Environments in Hilar Lymph Nodes of Cynomolgus Macaques

Chemokines provide critical immune cell homing and activation signals that if altered could affect the inflammatory milieu and cellular composition of lymphoid tissues. During HIV-1 and simian immunodeficiency virus (SIV)-infection, the virus triggers an increase in inflammation or activation, leading to immunodeficiency and development of opportunistic infections, such as in the lungs-a massive interface between the host and the environment. Chemokine, cytokine, and chemokine receptor expression profiles were determined using real-time reverse transcriptase-polymerase chain reaction and in situ hybridization in hilar lymph nodes (HiLNs) from cynomolgus macaques at different stages after infection with SIV/DeltaB670. Immunostaining of tissue sections and flow cytometric analysis of cryopreserved cells were used to examine cellular compositions of lymph nodes. Interferon-gamma, type 1 chemokine, and cognate chemokine receptor mRNAs were upregulated, whereas type 2 and homeostatic chemokine and chemokine receptor mRNAs were down-regulated in HiLNs after SIV infection. Local SIV and interferon-gamma levels were positively correlated with type 1 chemokine levels but negatively correlated with type 2 and homeostatic chemokine levels. Using in situ hybridization, Pneumocystis carinii rRNA was detected in lung-draining lymph nodes from animals with P. carinii pneumonia. Changes in the cellular composition of HiLNs included decreased proportions of CD4 cells and dendritic cells and increased proportions of CD8, CXCR3, and CCR5 cells. SIV infection of cynomolgus macaques dramatically alters the cellular homing signals of lung-draining lymph nodes, which correlated with changes in the immune cellular composition. These changes could contribute to the loss of immune function that defines AIDS.

Susceptibility to Mycobacterium tuberculosis Infection in HIV-Positive Patients Is Associated With CARD8 Genetic Variant

HLA and other genetic variants, playing an important role in innate and adaptive immunity, are known to influence tuberculosis (TB) development in HIV-1-positive (HIV+) patients. Because inflammasome genes contribute to HIV-1 susceptibility, we investigated the possible association between polymorphisms in inflammasome genes with HIV-1 and Mycobacterium tuberculosis coinfection (HIV+TB+) in a case/control cohort of Brazilian individuals. Nineteen single-nucleotide polymoprhims in 8 inflammasome genes (NLRP1, NLRP3, AIM2, CARD8, CASP1, IL1B, IL1R, and HSP90) were analyzed in HIV+TB+ Brazilian patients (from Recife, Pernambuco). CARD8 rs6509365 polymorphism was associated with HIV+TB+ (P = 5 × 10(-5)), suggesting a predisposing role of this variant in M. tuberculosis susceptibility in HIV+ subjects (odds ratio = 2.45). This effect is even strong when this allele is combined to CARD8 rs2043211 single-nucleotide polymoprhim. The results of this study support the novel association between CARD8 gene and HIV+TB+ coinfection, demonstrating that inflammasome genetics could influence HIV-1 infection and the development of opportunistic infection.

Analysis of the features of the course of HIV/AIDS in the Chernivtsi region

The research presents data about the clinical features and the spectrum of opportunistic infections in HIVinfected persons of the Chernivtsi region. An active involvement of the population into the epidemic process, a rapid clinical manifestation of asymptomatic HIV infection through the development of opportunistic infections are observed in the region. In most clinical cases a high rate of lesions of the skin, mucous membranes, the lymphatic, respiratory and central nervous systems was observed. A high percentage of co-infection of viral hepatites and HIV was fixed.

Assessment of ART adverse reactions and determinants at primary hospital in Ethiopia

Background: Much progress has been made in treating HIV infection in the last several years and currently antiretroviral therapy regimens are capable of reducing viral load of undetectable level with a consequent increase in T-lymphocyte, CD4+ counts and reduction in development of opportunistic infections. Hence, a substantial reduction in HIV associated morbidity and mortality can be attained. In spite of antiretroviral therapy benefits, adverse reaction to these drugs has been pointed to as one of the main reason for discontinuation, switch and non adherence to antiretroviral therapy. Methods: A cross-sectional retrospective review of patient record from December 2009-Novomber 2012 was performed to determine the common adverse drug reactions in patients taking ART medications. A sample of 154 who were taking ART medications at Ambo Zonal Hospital was studied and SPSS for windows software versions-16.0 was used for data analysis. Results: A total of patients with average age of 32.5 years who are taking ART drugs for more than 6 months were studied for the prevalence of adverse reactions. The frequency of GI tract adverse reactions were found to be 75 (48.7%) followed by CNS adverse effects, 55 (35.7%) skin reactions accounted for 29 (18.8%). The least frequently occurred adverse reactions were hematologic reaction (anemia). Patients with low BMI (OR =4.09, p =0.000), having comorbidities (OR=4.566, p =0.000), low CD4+, p =0.002) and treated by TDF/3-TC/EFV (OR=2.087, p =0.001) had high risk of developing adverse drug recreations. Conclusions: BMI, the presence of other diseases, types of regimen used, duration of therapy and CD4+ lymphocyte less than 400cell/mm 3 were strongly associated with the occurrence of adverse drug effects in this study.

DIAGNOSTIC VALUE OF THE IMMUNE TO ASSESS THE SEVERITY OF LYMPHOPROLIFERATIVE DISEASE, COMPLICATED BY AN OPPORTUNISTIC INFECTION

We have studied the results of the survey of cellular and humoral immunity in 260 patients lymphoproliferative diseases, complicated by the development of opportunistic infections. The study of the immunological characteristics of the cells allows you to set the source of origin of the pathological clone of the total diversity of morphologically similar forms limphoproliferativee disease.

Efectos indirectos de la infección por citomegalovirus

Despite improvements in prevention strategies, cytomegalovirus (CMV) continues to be the main cause of infection in solid organ transplant recipients. In these patients, in addition to direct effects, such as viral syndrome or invasive organ disease, CMV can cause indirect effects resulting from the interaction of the virus with the host's immune system. This interaction may increase immunosuppression, with a consequent rise in opportunistic infections and the risk of malignancies (Epstein-Barr virus-associated posttransplantation lymphoproliferative disease) and graft dysfunction. Currently, a direct causal relation between CMV and most of the indirect effects described cannot be established. However, numerous experimental and clinical studies have found an association between the development of these effects and CMV. Moreover, some of these effects, such as the development of opportunistic infections, have been reduced by CMV prophylaxis.

Silvestrol Modulates Direct Anti-Tumor Activity Against Epstein-Barr Virus (EBV)-Associated Lymphomas While Sparing Innate and Antigen Specific Adaptive Immunity

Abstract 104Epstein-Barr virus (EBV) is an oncogenic human herpes virus that infects more than 90% of people worldwide and is associated with a broad spectrum of malignant lymphoproliferative disorders (EBV-LPD), and nasopharyngeal and gastric carcinomas. Chemotherapy often leads to prolonged immune suppression, development of opportunistic infections, including EBV reactivation, and increased risk of relapsed disease. The poor prognosis of EBV+ diseases makes it essential to identify novel agents that can deliver direct anti-tumor activity while preserving innate and EBV-specific adaptive immune surveillance.Silvestrol is a cyclopenta[b]benzofuran derived from the plant genus Aglaia and has been shown to possess potent anti-tumor activity against hematologic and solid tumors. Silvestrol interferes with the translation of mRNA with complex 5’ untranslated regions often found on pro-survival oncoproteins. Silvestrol exhibits anti-tumor activity against malignant B-cell lines while causing minimal toxicity to peripheral blood mononuclear cells (PBMC) and resting T cells. To examine the potential selective anti-tumor activity and functionally address the effects of silvestrol on adaptive and innate immune function, we utilized in vitro and in vivo EBV lymphomagenesis models. Fully transformed EBV+ Lymphoblastoid lines (LCL) were derived from EBV-LPD tumors of severe combined immune deficient (SCID) mice engrafted with PBMC from EBV-positive donors (hu-PBL-SCID). EBV-LCLs were plated in the presence of silvestrol (2 – 50nM) and proliferation (MTS assay) and apoptosis (Annexin V/PI) was evaluated (24, 72, 120hr). While silvestrol showed potent anti-proliferative activity at these concentrations, we observed minimal cell death (IC50 = 10nM). The anti-proliferative activity of silvestrol was associated with loss of LMP-1 expression, an EBV oncogene essential for B-cell transformation. Examination of LMP1 induced pathways showed decrease in pAkt levels and an increase in NFkB/p65 levels (total and phospho).To examine the functional consequences of silvestrol on immune surveillance, we utilized a co-culture system where EBV+ LCLs are plated in the presence of autologous PBMCs. Autologous LCL/PBMC co-cultures were plated with silvestrol or DMSO vehicle control and allowed to incubate for 10 days. When EBV+ LCLs are irradiated, expansion of memory EBV-specific CD3+/CD8+ cytotoxic T cells (CTLs), capable of cytotoxicity and IFNg production, is observed. The addition of silvestrol (2–10nM) to co-cultures did not hinder CTL expansion or IFNg production. When unirradiated LCLs were plated in co-cultures, CTL expansion was seen, however, EBV+ LCLs became the dominant population in control conditions by day 10 of culture. Addition of silvestrol to unirradiated co-cultures (2–10nM) led to marked expansion of memory CD3/CD8+ CTLs as well as CD56br NK cells. A dose dependent ablation of viable EBV-LCLs was observed in unirradiated co-cultures supporting the notion that silvestrol allowed for the expansion of both innate and adaptive immune effectors that were capable promoting anti-tumor activity. Immune effector populations that expanded in the presence of silvestrol showed preservation of direct cytotoxicity (adaptive immunity) and antibody dependent cell-mediated cytotoxicity assays (ADCC) against EBV+ LCLs (innate immunity) that was comparable to untreated effector populations.We next examined the efficacy of silvestrol in vivo using the Hu-PBL SCID model depleted of murine NK cells. On d14, mice were randomized to receive treatment with either silvestrol (1.5mg/kg) or vehicle control by IP injection every 48hr. Human cell engraftment was measured by quantifying human IgG in blood. While silvestrol treatment did not affect cell engraftment, all treated mice showed improved survival when compared to mice treated with vehicle control (100% silvestrol vs. 22% control alive at day 140, p ≤0.001). Control mice showed decreased body weight (85% decrease) and splenomegally (spleen wt 421.6 mg vs 72.9 mg for control vs silvestrol) on necropsy at day 140, p≤.0004). There were no toxicities observed in silvestrol treated mice. Low dose silvestrol promotes direct anti-proliferative activity of EBV-transformed LCLs while sparing antigen specific adaptive and innate immune effector function that is capable of delivering potent anti-tumor activity in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

HIV pathogenesis and cytokine secretion patterns in AIDS-related opportunistic infections and HIV-1/HBV co-infections

HIV infection, a disease caused by the human immunodeficiency virus (HIV), is rapidly becoming one of the greatest public health challenges faced by medical doctors, public health experts, implementers, biomedical scientists, governments, medical institutions, nongovernmental organisations, as well as communities and the vulnerable population in any one society, such as, the women and children. HIV-1 infection is characterised by chronic immune activation which often results in impaired effector functions, such as alterations in cytokine production, which may contribute to the development of opportunistic infections especially during the late stage of the disease. Co-infection with hepatitis b virus (HBV) is very common among hiv-1 infected patients due to their common routes of transmission. However, studies elucidating the interactions between HBV, HIV-1 and the immune system are not very common and as such a lot remains undiscovered. a host of cytokines both proand anti-inflammatory cytokines; have been implicated in the pathogenesis of HIV disease and in HBV/HIV-1 co-infections. However, in most cases it has been quite difficult to allocate distinct roles to specific cytokines, due to the fact that cytokines are pleiotropic in nature and have a huge tendency to interchange roles. Therefore, further studies will be required in order to correlate specific cytokine secretion patterns with specific opportunistic infections, and HIV-1/HBV co-infections. These will in-turn enable scientists to further understand the existing relationships between HIV-1 and the immune system as well as interactions between HIV-1 and HBV.

Emergence ofHaemophilus influenzaeStrains in the Nasopharynx of Children with Tuberculosis

Being rigorously studied, epidemiology aspects of Haemophilus influenzae carriage are still remaining unclear. Especially it concerns such a group as children with low immune status and also such group as children with tuberculosis infection. We examined nasopharyngeal tract of children with tuberculosis infection in remission and checked how often these children are carriers of Haemophilus influenzae strains. Also we gained microbiology characteristics of the isolates and defined the clinical significance of H. influenzae carriage in development of opportunistic infections in children with the tuberculosis infection.

Major lupus organ involvement: severe lupus nephritis

Lupus nephritis is a common and severe complication of systemic lupus erythematosus. A number of patients have nephritis as a presenting feature that, in its severe form, can shortly lead to end-stage renal disease and/or death. Renal flare usually occurs a few years after the first episode and is remarkably predominant in the Asian population. Frequent monitoring for renal flare enhances early recognition and timely treatment. The mainstay therapy continues to be the prolonged use of cytotoxic/immunosuppressive drugs that have a number of undesirable effects, particularly ovarian failure and development of opportunistic infections. This review will focus on the pathogenesis and the unique genetic factors found in Asian patients with lupus nephritis. Here, we propose an appropriate management scheme for the treatment of lupus nephritis in Asian patients.

Factors associated with development of opportunistic infections among patients on ART at a Ugandan Program-MJAP

Results Data of 4,878 patients on ART was analyzed. 3,247 (67.2%) were females. Median age 33 years (SD 8.5) and Median weight 53 kg (SD 10.0). Incidence of OIs 329 (6.7%); Oral candidiasis 106 (32%), Vaginal candidiasis 80 (24%), Kaposi’s sarcoma 40 (12%), Herpes simplex 28 (9%), Oesophageal candidiasis 25 (8%), Tuberculosis 15 (5%), others 38 (10%). WHO stage 3&4 (OR 2.64, CI 2.04-3.43, P < 0.001), CD4 count <100 cells/mm (OR 1.92, CI 1.52-2.42, P < 0.001), not being married (OR 1.69, CI 1.33-2.14, P < 0.001) and having no formal education (OR 1.61 CI 1.09-2.36, P = .0015) were significantly associated with development of OIs among patients on ART. Sex, age, body weight, ART regimen and Karnofsky score were not associated with development of OIs.

Conventional splenic dendritic cells regulate development of graft-versus-host disease. (145.10)

Abstract The use of allogeneic hematopoietic stem cell transplant (HSCT) to treat hematologic malignancies is associated with the development of graft-versus-host disease (GVHD), a frequently fatal complication that limits successful outcomes. The treatment of GVHD often requires potent immunosuppressive drugs and can predispose to relapse of the original cancer and the development of opportunistic infection. Understanding how GVHD is initiated may help to identify targets for therapeutic interventions that do not require wholesale suppression of the immune response. Prior studies have shown that host antigen presenting cells (APCs) are critical to GVHD induction. We examined how functionally distinct host splenic DC subpopulations (CD4+ vs. CD8+) regulate the development of GVHD mediated by donor CD4+ T cells. When compared to purified Balb/c CD4+ DCs, we found that Balb/c CD8+ DCs significantly increased C57BL/6 CD4+ T cell proliferation and cytolytic activity in vitro. To control the source of T cell stimulation for in vivo HSCT studies, we created chimeric Balb/c mice that have C57BL/6 APCs (C57BL/6 → Balb/c). Subsequently, we found that transplantation of C57BL/6 CD4+ T cells stimulated ex vivo by CD8+ Balb/c DCs resulted in more robust GVHD (survival, clinical score, pathology) compared to T cells activated by CD4+ DCs. Targeting specific DC-T cell interactions early in HSCT may help to control the severity of GVHD without requiring wholesale suppression of the immune system.

HIV-1 infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs.

HIV causes a chronic infection characterized by depletion of CD4+ T lymphocytes and development of opportunistic infections. Despite drugs that inhibit viral spread, HIV has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy and the immune response. Here we used CD34+ cells from infected people as well as in vitro studies of wild type HIV to demonstrate infection and killing of CD34+ multipotent hematopoietic progenitor cells (HPCs). In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A novel reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have important implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

Opportunistic infections and organ‐specific diseases in HIV‐1‐infected children: a cohort study (1990–2006)

Highly active antiretroviral therapy (HAART) has dramatically changed the natural history of HIV infection in children, but there are few studies in the literature about the incidence of clinical manifestations after HAART in this population, compared with adults. The aim of this study was to describe the influence of the widespread use of HAART on the development of opportunistic infections and organ-specific diseases in HIV-infected children. An observational study of a cohort of 366 vertically HIV-infected children followed from 1990 to 2006 was carried out. According to the main antiretroviral protocol used, three calendar periods (CPs) were defined and compared: CP1 (1990-1996: no patients on HAART), CP2 (1997-1999: <60% on HAART) and CP3 (2000-2006: >60% on HAART). Children experienced a progressive increase in CD4 T cell count (P<0.05) and a decrease in HIV viral load from 1996 onwards (P<0.05). Similarly, rates of death, AIDS, opportunistic infections (bacteraemia, candidosis, cryptosporidiosis and bacterial pneumonia) and organ-specific diseases (wasting syndrome, thrombocytopenia, cardiomyopathy, lymphoid interstitial pneumonia and HIV-associated encephalopathy) were lower in CP2 and CP3 than in CP1. This study provides evidence of improved clinical outcomes in HIV-infected children over time and shows that mortality, AIDS, opportunistic infections and organ-specific diseases declined as HAART was progressively instituted in this population.

Successful prophylaxis with valaciclovir for relapsing HSV-1 in a girl treated with infliximab for moderate Crohn's disease

Biological agents such as inhibitors of tumour necrosis factor alpha (TNF-alpha ) are associated with the development of opportunistic infections. Although there are no international recommendations for the management of opportunistic infections, their prevention is a key safety issue for patients with inflammatory bowel disease (IBD). We report that chemoprophylaxis with oral valaciclovir was effective in preventing Herpes simplex virus (HSV-1) reactivation in a girl treated with infliximab for Crohn's disease.

254 Factors associated with development of Opportunistic Infections among patients on ART in Uganda

254 Factors associated with development of Opportunistic Infections among patients on ART in Uganda

Combined tumor necrosis factor-α (TNF–α) and interleukin-2 (IL-2) blockade in acute steroid refractory graft-versus-host disease (SR-GVHD) following allogeneic hematopoietic stem cell transplantation (HCT)

7023 Background: SR-GVHD is a frequent and often fatal complication of HCT. A variety of inflammatory cytokines have been implicated in the pathogenesis of GVHD; single agent therapy against targets such as the IL-2 receptor α chain (daclizumab) or TNF-α (infliximab) has modest activity in SR-GVHD. We hypothesized that concomitant blockade of both TNF-α and IL-2 pathways would be more effective in controlling SR-GVHD than inhibition of either cytokine alone. Methods: The incidence of and outcome following SR-GVHD in 141 pts undergoing nonmyeloablative HCT from an HLA-matched family donor at our institution between February 2001 and November 2008 were analyzed. All SR-GVHD pts were treated with a combination of daclizumab (1 mg/kg, days1, 4, 8, 15, 22) and infliximab (10 mg/kg, days1, 8, 15, 22); in addition, aspergillus prophylaxis, empiric broad-spectrum antibiotics, and a rapid reduction in the dose of corticosteroids was initiated in order to minimize the risk of opportunistic infections associated with immunosuppression. Results: Twenty-three pts (23/141, 16%) developed SR-GVHD (median age 35 y, range 17–65 y); involved organs included the GI tract (n = 23), liver (n = 3), and skin (n = 8). We observed a remarkably high response rate following therapy, with 20/23 (87%) pts experiencing complete resolution of GVHD. Responses were usually delayed (median onset 2 weeks) but durable. The most notable complication associated with therapy was the development of opportunistic infections (invasive fungal infections in 3 pts); in 2/3 of these cases, prophylactic antifungal therapy had been discontinued prematurely due to drug toxicity. The median survival for the SR-GVHD cohort was 255 days (range 67–2,148 days), with 10/23 pts surviving at the time of this analysis. Causes of death included underlying cancer (5 pts), CMV disease (2 pts), and infectious complications (5 pts). Conclusions: These data suggest combined TNF-α /IL-2 blockade is a highly effective therapeutic option for pts with SR-GVHD and highlight the need for aggressive antimicrobial prophylaxis in the management of this condition. No significant financial relationships to disclose.

Varicella Zoster Meningitis in a Pregnant Woman with Acquired Immunodeficiency Syndrome

Between 6000 and 7000 women in the United States infected with human immunodeficiency virus (HIV) give birth annually. It is well known that HIV-related immunosuppression significantly increases the risk for acquiring opportunistic infections (OIs). However, there is limited information regarding the relationship of pregnancy in the setting of HIV/AIDS infection, subsequent development of OIs, and maternal and fetal outcomes. A pregnant 36-year-old woman with AIDS was diagnosed with varicella zoster meningitis. Weight-based therapy with acyclovir was initiated with clinical improvement in symptoms. Care of a pregnant HIV-infected patient with an OI poses a unique diagnostic and therapeutic challenge for clinicians. Early diagnosis and initiation of appropriate treatment may provide an opportunity to improve both maternal and fetal outcomes.

Chronic Disseminated Mycobacterium xenopi Infection in a Cat with Idiopathic CD4+ T Lymphocytopenia

A 3-year-old castrated male domestic shorthair cat was referred for examination in August 2000 with a 3-week history of poor appetite, vomiting, and weight loss. One week before referral, evaluation by the referring veterinarian identified anemia (PCV 21% [reference range 29–48%]) and moderate renal insufficiency (BUN 55mg/dL [reference range 14–36mg/dL], serum creatinine 4.2mg/dL [reference range 0.6–2.4mg/dL], urine specific gravity 1.015) with an inflammatory urine sediment (proteinuria, white blood cells, bacteria) on a voided sample. The results of enzyme-linked immunosorbent assays for feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies were negative. Radiographs disclosed bilateral renomegaly and microcardia. Abdominal ultrasound examination identified a thin cortex in the left kidney and both kidneys were hyperechoic. The cat was treated with lactated Ringer’s solution IV and enrofloxacin 6mg/kg/d IV for 3 days. Upon examination at the referral hospital, the cat was quiet and responsive. Physical examination abnormalities were limited to bilaterally enlarged kidneys that appeared to be painful on palpation and a body condition score of 3/9. Body weight was 3.6 kg. On CBC there was a severe nonregenerative macrocytic normochromic anemia (PCV 17% [reference range 30–47%], MCV 55 fL [reference range 41–51 fL], MCHC 31 g/dL [reference range 31–35 g/dL], aggregate reticulocytes 20,880/mL) in the presence of normal serum erythropoietin concentration (17mU/mL [reference range 10–30mU/mL]). The WBC count (10,200/mL [reference range 5,500–19,500/ mL]), differential cell count (segmented neutrophils 7,040/mL [reference range 2,500–12,500/mL], lymphocytes 2,860/mL [reference range 1,500–7,000/mL], monocytes 260/mL [reference range 0–800/mL], eosinophils 50/mL [reference range 0–1,500/mL]), and platelet count (485,000/mL [reference range 300,000–800,000/ mL]) were normal. Serum chemistry results indicated moderate azotemia (BUN 36mg/dL [reference range 16–34mg/dL], serum creatinine 3.5mg/dL [reference range 0.7–2.3mg/dL]), increased AST activity (173U/L [reference range 5–30U/L]), and hypoalbuminemia (1.8 g/dL [reference range 2.0–3.1 g/dL]). Results of urinalysis collected by cystocentesis indicated isosthenuria (USG 1.011), proteinuria, and bacteruria. Aerobic urine culture was negative, and plasma prothrombin and partial thromboplastin times were within normal limits. Ultrasonographic examination of the abdomen identified moderate bilateral renomegaly with pyelectasia, mesenteric lymphadenopathy, and an enlarged hypoechoic pancreas. By means of ultrasound guidance, fineneedle tissue aspirates were collected from kidney, mesenteric lymph node, and pancreas, and slides were prepared for cytologic evaluation using Wright-Giemsa stain. Preparations obtained from the kidney were very cellular, consisting primarily of large, epitheliod macrophages arranged in sheets and individually. The cytoplasm of many of the macrophages contained long, thin, negatively staining, rod-shaped inclusions consistent withMycobacteria spp. (Fig 1). Renal epithelial cells were also observed in the preparation.Macrophages containing similar negatively staining rods were observed in tissue aspirate preparations from the pancreas and a mesenteric lymph node, along with pancreatic epithelium and lym-

CD52 Expression in Adult Acute Lymphoblastic Leukemia (ALL): Quantitative Flow Cytometry Provides New Insights.

We recently tested the feasibility of incorporating Alemtuzumab, a humanized anti-CD52 monoclonal antibody, into frontline therapy of adult ALL (CALGB 10102) in an attempt to eradicate minimal residual disease (MRD). We have previously reported that CD52 expression occurs in 68% of newly diagnosed ALL. This assessment was based upon the use of a qualitative flow cytometric assay performed on all pre-treatment cases in a central CALGB reference laboratory. Cases with > 10% CD52 expression on lymphoblasts relative to an isotype control were considered CD52 positive and eligible to receive Alemtuzumab during post-remission therapy. To better characterize the CD52 expression level on lymphoblasts and to obtain insights for the design of future subset specific therapies in adult ALL, we developed a quantitative assay to measure CD52 antigen density on lymphoblasts using custom PE conjugated clinical grade Alemtuzumab on 29 cases of precursor B-cell (pre-B) ALL and 9 cases of precursor T-cell (pre-T) ALL. In this assay, CD52 is expressed in arbitrary units of antibody bound per cell (ABC). We also measured CD52 levels on residual normal B and T lymphocytes in every specimen to calculate a normalized ratio (NR) of lymphoblast CD52ABC/ normal lymphocytes CD52 ABC. The results are tabulated below:CD52 ExpressionCell TypePrecursor B-Cell ALL (n=29) median (range)Precursor T Cell ALL (n=9) median (range)CD52 ABC unitsBlasts27658 (2042–206952)10222 (3784–35337)CD52 ABC unitsNormal B Lymphocytes125475 (59872–282245)146478 (68098–291878)CD52 ABC unitsNormal T Lymphocytes64142 (27406–141635)78720 (22412–144350)CD52 NRBlasts/Normal B Lymphocytes0.19 (0.02–0.88)0.06 (0.02–0.16)CD52 NRBlasts/Normal T Lymphocytes0.38 (0.04–2.16)0.12 (0.07–0.26)Pre-B lymphoblasts express significantly lower levels of CD52 antigen than normal B lymphocytes (p<0.0001). Similarly, Pre-T lymphoblasts express significantly lower levels of CD52 antigen than the normal T lymphocytes (p<0.0001). Moreover, lymphoblasts in pre-T ALL show a near-significant lower expression of CD52 than pre-B lymphoblasts (two-sided p=0.05). Calculation of the NR confirms these observations. We have also begun to investigate CD52 expression by cytogenetic subset. Of note, we have determined that Philadelphia chromosome positive (Ph+) lymphoblasts were CD52+ in 26/28 (93%) cases studied; quantification of CD52 expression in Ph+ cases and in other common cytogenetic subsets is in progress. Correlation of CD52 expression with Alemtuzumab plasma levels and with development of opportunistic infections is underway. In conclusion, these data are the first to demonstrate that lymphoblasts from patients with untreated ALL express lower levels of CD52 antigen than residual normal lymphocytes present at the time of diagnosis. These findings may lead to improved selection of patients most likely to benefit from administration of Alemtuzumab and may have important implications for optimizing dosing and scheduling of Alemtuzumab during ALL therapy.