Michael C. Sneller

Inherited Human Caspase 10 Mutations Underlie Defective Lymphocyte and Dendritic Cell Apoptosis in Autoimmune Lymphoproliferative Syndrome Type II

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.

Cyclophosphamide-induced cystitis and bladder cancer in patients with Wegener granulomatosis.

Wegener granulomatosis is a necrotizing granulomatous vasculitis that typically involves the upper and lower respiratory tract and the kidneys. Standard therapy for Wegener granulomatosis includes the daily administration of low-dose oral cyclophosphamide and corticosteroid therapy [1, 2]. This therapeutic regimen has dramatically improved the survival of patients with this otherwise fatal disease: More than 90% of patients treated with cyclophosphamide and corticosteroid therapy improve markedly, and 75% achieve complete remission of disease [1, 3]. However, extended follow-up of patients with Wegener granulomatosis indicates that relapse of disease is common, and repeated and prolonged courses of cyclophosphamide can be associated with serious long-term toxicities, including bone marrow suppression, infertility, hemorrhagic cystitis, and the development of cancer [3]. Hemorrhagic cystitis and bladder cancer are well-recognized complications of cyclophosphamide therapy for both malignant [4] and nonmalignant diseases [3, 5-7]. However, the relations among total cyclophosphamide dose, the development of cystitis, and the occurrence of bladder cancer have not been well defined. In this report, we describe the incidence, clinical manifestations, and natural history of cyclophosphamide-mediated urotoxicity in a cohort of patients with Wegener granulomatosis. We identify risk factors associated with the development of cyclophosphamide-induced bladder cancer and discuss recommendations for surveillance. Methods Patients From 1967 to 1993, 145 patients with Wegener granulomatosis were treated with cyclophosphamide at the Warren G. Magnuson Clinical Center of the National Institutes of Health (NIH). The clinical features of the underlying disease in all but 3 of these patients have been reported previously [3]; the clinical and demographic characteristics of these patients are summarized in Table 1. Individual patients were followed for 0.5 to 27 years (median, 8.5 years), for a total of 1333 patient-years. Table 1. Demographic and Clinical Characteristics of 145 Patients with Wegener Granulomatosis Who Were Treated with Cyclophosphamide* Treatment Protocol We used the standard cyclophosphamide treatment regimen, which has been described previously [1, 3]. Therapy consisted of 1) oral cyclophosphamide, 2 mg/kg of body weight per day and 2) prednisone, 1 mg/kg of body weight per day. If patients improved substantially after the first month of treatment, the prednisone dose was gradually tapered to an alternate-day regimen, and prednisone therapy was eventually discontinued. Cyclophosphamide therapy was continued for at least 1 year after patients achieved complete remission. The cyclophosphamide dose was then tapered by 25-mg decrements of therapy every 2 to 3 months until discontinuation of therapy or until disease recurrence required an increase in dose. The cyclophosphamide dose was adjusted as needed to keep the absolute neutrophil count higher than 1.0 106/L. If substantial toxicity required the permanent discontinuation of cyclophosphamide therapy and if signs of active vasculitis were present, azathioprine, chlorambucil, or (after 1990) low-dose weekly methotrexate therapy was allowed. Eight patients with fulminant disease initially received intravenous cyclophosphamide at daily doses of 3 to 5 mg/kg. When their disease stabilized, these patients were switched to the standard oral cyclophosphamide regimen. Fourteen patients received monthly pulses of intravenous cyclophosphamide, 1 g/m2 body surface area; 13 of these 14 patients also received one or more courses of the standard oral cyclophosphamide regimen. Thus, 144 of the 145 patients received oral cyclophosphamide therapy for some period of time. Patients were evaluated at the NIH every 1 to 3 months. Those who achieved remission of disease and maintained it for 1 year were subsequently seen every 6 months. For each patient, urinalysis was done at every visit, and a cytologic examination of urine was done every 6 to 12 months. All patients received cyclophosphamide as part of clinical research protocols approved by the National Institute of Allergy and Infectious Diseases (NI-AID) Institutional Review Board, the NIAID Clinical Director, and the director of the NIH Clinical Center. All patients gave written informed consent. Urine Cytology Cytologic examination of urine was done at each evaluation. Sediments obtained from voided urine specimens were fixed in Saccomanno solution (Lerner Laboratories, Pittsburgh, Pennsylvania), immobilized on membrane filters (Millipore, Chicago, Illinois) or by cytospin, placed in 95% ethanol, and stained with Papanicolaou stain. Urine samples obtained as much as 6 months before each cystoscopic examination were reviewed retrospectively and were correlated with subsequent bladder biopsy specimens. Cellular cytologic features were placed in the following categories according to the following criteria [8-10]: 1. Negative: no important epithelial abnormalities. 2. Atypia: some nuclear abnormalities in epithelial cells, but the changes could not be definitely placed in categories 3, 4, or 5. 3. Therapeutic or viral: cytologic changes consistent with polyomavirus infection or cyclophosphamide toxicity. In the absence of diagnostic inclusions, the two types of changes are indistinguishable, and we therefore grouped them together. The nuclear enlargement and hyperchromasia associated with polyomavirus or chemotherapeutic effect should be distinguished from high-grade dysplasia or carcinoma. 4. Dysplasia or possible low-grade transitional-cell carcinoma: a few cells in a voided urine sample, either singly or in clusters, that have slightly enlarged, irregular nuclei with increased granularity in chromatin distribution and small or absent nucleoli. 5. Transitional-cell carcinoma: high-grade lesions meeting unequivocal criteria of malignancy. Definition of Terms Nonglomerular hematuria was defined as microscopic or gross hematuria not associated with the presence of erythrocyte casts or declining renal function. Glomerulonephritis causing hematuria associated with erythrocyte casts (glomerular hematuria) occurred at least once in 116 of the 145 patients (Table 1). If hematuria persisted after the treatment of glomerulonephritis and the disappearance of erythrocyte casts, or if hematuria not associated with the presence of erythrocyte casts ever developed, patients were considered to have nonglomerular hematuria and had cystoscopy (see below). Cyclophosphamide-induced cystitis was defined as nonglomerular hematuria associated with characteristic cystoscopic bladder changes. These changes included patchy areas of neovascularity and telangiectasia manifested as an increased number of tortuous, thin-walled veins and small areas of hemorrhage in or under the bladder epithelium. The mucosa between the hypervascular areas may appear normal or pale with decreased vascularity. Cystoscopy Cystoscopy was done to evaluate nonglomerular hematuria (microscopic or gross) in patients receiving cyclophosphamide. Only the results of cystoscopies done at the NIH Clinical Center by members of the Urologic Oncology Section of the National Cancer Institute are included in this report. Most of these cystoscopies were done by two of the authors; all cystoscopy results were reviewed by these two authors. Random biopsies were done only if the results of cytologic examination of urine suggested malignancy. Patients having cystoscopy had intravenous pyelography or retrograde pyelography, or both, at least once to evaluate the upper urinary tract. Statistical Analysis The frequencies of clinical findings were compared by using the Fisher exact test for association with bladder cancer and nonglomerular hematuria; adjustments were made for multiple comparisons of microscopic and gross hematuria with bladder cancer using the modified Bonferroni method [11]. The effects of fixed covariates (sex, history of smoking, age at onset of Wegener granulomatosis disease, and duration of disease before first cyclophosphamide treatment) and time-varying covariates (microscopic hematuria, gross hematuria, total cyclophosphamide dose, and duration of cyclophosphamide therapy) on the development of bladder cancer were examined using Cox proportional-hazards regression analysis [12, 13]. Medians and other percentiles for variables dependent on follow-up time were estimated by using the Kaplan-Meier method [14]. The cumulative distributions determined by the Kaplan-Meier method were compared with the log-rank test. Risk estimates for the development of bladder cancer in patients with Wegener granulomatosis who were treated with cyclophosphamide were determined by comparing observed rates with the expected rates for the United States population, which were obtained from the Surveillance, Epidemiology, and End Results (SEER) Cancer Statistics Review, 1973-1991 [15]. Results Hematuria Seventy-three of 145 patients treated with cyclophosphamide (50%) developed nonglomerular hematuria; the median time to development for all patients was 37 months of receipt of cyclophosphamide (95% CI, 32 to 55 months), and the median dose before development was 124 g (CI, 82 to 149 g) (Table 2 and Figure 1). Forty-one patients (56%) presented with microscopic hematuria; 32 (44%) presented with gross hematuria. Twenty-eight patients had more than one recurrent episode of nonglomerular hematuria (microscopic or gross), sometimes years after cyclophosphamide therapy had been discontinued. Eighteen of the 73 patients developed hematuria after cyclophosphamide therapy was discontinued. Manifestations of active vasculitis necessitated that cyclophosphamide therapy be continued in 26 of the 55 patients who developed nonglomerular hematuria while receiving this therapy. Table 2. Clinical Characteristics of the 73 Patients Treated with Cyclophosphamide Who Developed Nonglomerular Hematuria Figure 1. Cumulative r

An Inherited Disorder of Lymphocyte Apoptosis: The Autoimmune Lymphoproliferative Syndrome

Dr. Stephen Straus (Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health [NIH], Bethesda, Maryland): The autoimmune lymphoproliferative syndrome (ALPS) is a recently defined illness that arises in early childhood and can have fatal complications (1-8). It is associated with prominent nonmalignant lymphadenopathy, hepatosplenomegaly, and autoimmune manifestations. Underlying ALPS are heritable mutations in genes that regulate lymphocyte survival by triggering programmed death of lymphocytes, or apoptosis. More important than the mere description of ALPS, however, are the novel insights that this description affords into the mechanisms that regulate lymphocyte homeostasis and contribute to autoimmunity. Historical Perspective Selected features of ALPS have been recognized for decades, but the full-blown syndrome is rare and has only recently been appreciated. Several authors have described families with significant adenopathy and splenomegaly (9-13) and other families with hemolytic anemia, thrombocytopenia, or neutropenia in association with circulating autoantibodies (14-20). Splenomegaly is a feature of autoimmune diseases (such as the Felty syndrome), and moderate lymphadenopathy is seen in up to 70% of patients with lupus (21). The constellation of lymphadenopathy, splenomegaly, and autoimmune cytopenia, however, was described by Canale and Smith in 1967 (22). Weisdorf and Krivit (23) and others (24, 25) noted that similar patients had decreased proportions or function of lymphocyte subsets. Contemporary evaluation of some of these older cases, including a family followed at the NIH, has shown them to be cases of ALPS (4, 13; Straus SE. Unpublished data). Clues to the nature of some of these familial diseases emerged in the early 1990s, when we realized that affected patients resembled mice with the MRL/lpr phenotype; these mice exhibit progressive lymphoproliferation and autoantibody-mediated renal and vascular disease (1, 26, 27). Moreover, the usually rare subset of T cells that show neither the CD4 nor the CD8 co-receptors (CD3+ CD4 CD8 ), or double-negative cells, circulates in increased numbers both in these mice and in our patients. In 1989, Trauth and coworkers (28) reported that a protein called Apo-1 triggered apoptosis of lymphocytes. In 1992, Watanabe-Fukunaga and colleagues (29) found that lpr mice failed to express that same antigen, which they called Fas and, later, CD95. In 1994, the APT1 gene encoding the human homologue of murine Fas was cloned (30). Shortly thereafter, Rieux-Laucat and associates in France (2) and Fisher and colleagues at the NIH (3) demonstrated defective apoptosis and specific Fas mutations in eight children with ALPS. In the ensuing years, more than 40 similar patients have been described (4-8). The autoimmune lymphoproliferative syndrome (sometimes called the Canale-Smith syndrome [4]] represents a failure of apoptotic mechanisms that help maintain normal lymphocyte homeostasis, with a consequent accumulation of lymphoid mass and persistence of autoreactive cells. It is operationally defined as chronic, nonmalignant lymphoproliferation in patients with 1) an elevated percentage [>1%] of double-negative T cells and 2) defective lymphocyte apoptosis that produces a characteristic, if not pathognomic, pathologic picture on microscopic section of the lymph node or spleen (31). Autoimmunity is evident at some point in almost all affected patients. Most cases of ALPS are associated with specific Fas mutations, and yet-undefined mutations in other apoptosis genes are thought to underlie cases in patients with normal Fas. One remarkable case from our clinic shows many features of ALPS (Figure 1). Figure 1. Clinical, radiologic, and histologic features of patients with the autoimmune lymphoproliferative syndrome. A. B. C. D. E. F. G. Case History At age 18 months, NIH ALPS patient 2 was noted to have adenopathy, splenomegaly, and anemia (1, 3, 6). She underwent splenectomy at age 2 years for refractory anemia. Biopsies of splenic tissue, liver, lymph node, and bone marrow were nondiagnostic. Clinical photographs (Figure 1 A) and computed tomographic scans (Figures 1F and 1G) confirmed the presence of persistent, massive enlargement of all lymph node chains and organomegaly. Laboratory studies showed continuing anemia, polyclonal gammopathy, and positive results on a direct Coombs test. Autoimmune manifestations have included glomerulonephritis at age 2 years, idiopathic thrombocytopenic purpura (ITP) at age 9 years, and autoimmune biliary disease since age 10 years. Lymphocyte phenotyping showed absolute increases in B-cell and T-cell counts with a polyclonal expansion of T cells, 25% of which were double negative. The lymph nodes showed lymphoid hyperplasia and plasmacytosis (Figure 1 B), and more than 50% of T cells were double negative (Figures 1C, 1D, and 1E). In vitro studies of peripheral blood mononuclear cells showed defective Fas-mediated apoptosis, and genomic sequencing identified a point mutation in the APT1 gene encoding Fas. Clinical Features and Management Dr. Michael C. Sneller (Laboratory of Immunoregulation, NIAID, NIH): Between 1990 and 1997, the NIH evaluated 45 patients under approved protocols for unexplained chronic lymphadenopathy, splenomegaly, or both. Among these patients, ALPS was subsequently diagnosed in 26 (12 male and 14 female). The remaining patients had some features of ALPS or had entirely different conditions. The Table shows the clinical and salient laboratory features of these 26 patients, of whom 23 had documented Fas mutations. The clinical and laboratory features of ALPS did not differ between patients who had mutations and those who did not. Clinical data on 9 of these patients are reported elsewhere (1, 6). Table. Clinical and Immunologic Features in 26 Patients with the Autoimmune Lymphoproliferative Syndrome Lymphoproliferative Disease All 26 patients initially presented because of lymphadenopathy or splenomegaly at a median age of 11.5 months (range, 1 month to 9 years). Most had both splenomegaly and lymphadenopathy. Splenomegaly was frequently of massive proportions, and hepatomegaly was also common (Figure 1 G). Sixteen patients underwent splenectomy, most often for severe hypersplenism. Lymphadenopathy was massive and distorted normal anatomic landmarks in some patients (Figure 1 A). Enlargement of abdominal and thoracic lymph nodes was frequently seen on computed tomography (Figure 1 F). Regardless of its extent, lymphadenopathy persisted for 2 or more years in almost all patients. Histopathologic analyses of lymph nodes from patients with ALPS show architectural preservation, florid reactive follicular hyperplasia, and marked paracortical expansion with immunoblasts and plasma cells (1, 6, 31) (Figure 1 B). The paracortical expansion may be extensive enough to suggest a diagnosis of immunoblastic lymphoma, with many cells expressing the Ki-67 antigen indicative of active proliferation (32). However, the tissues show no chromosomal abnormalities or evidence of clonality (31). Increased numbers of double-negative T cells are also seen in the paracortical region of lymph node tissue (Figures 1C, 1D, and 1E). This combination of follicular hyperplasia and paracortical expansion by a mixed polyclonal infiltrate containing double-negative T cells differentiates ALPS from other benign and malignant lymphoproliferative lesions. Autoimmunity Circulating autoantibodies, overt autoimmune disease, or both were found in 23 of the 26 patients. Potentially pathogenic autoantibodies were detected in 22 patients (Table) but were not always associated with disease. For example, the direct Coombs test detected antibodies to erythrocytes in 19 patients, but 6 of these patients had no evidence of hemolysis. At least one autoimmune disease was documented in 17 patients (Table) and was evident in 4 of the 17 at the time of initial presentation with lymphoid hyperplasia. In the remaining 13 patients, autoimmune disease developed 6 months to 17 years later, suggesting that the proportion of patients with autoimmune disease increases over time. The most common autoimmune diseases were hemolytic anemia and ITP (Table). Nine patients had at least one episode of hemolysis during which hemoglobin levels decreased to less than 4.4 mmol/L (7 mg/dL). In seven of eight patients with ITP, platelet counts decreased to less than 20 109 cells/L. Five patients also had hemolytic anemia, either concomitantly with active ITP or as isolated episodes with normal platelet counts. Neutropenia (absolute neutrophil count<1.0 106 cells/L) in six patients seemed to result from autoimmune mechanisms because it developed after splenectomy and in the setting of normal myeloid cellularity on bone marrow examination. Several nonhematologic autoimmune diseases also occurred in this group of patients. One patient developed the Guillain-Barr syndrome. Of two patients with glomerulonephritis, one later developed ITP and autoimmune biliary disease (Figure 1 A). Thus, in ALPS, multiple autoimmune diseases involving different organ systems may occur over time in a single patient. No patient developed opportunistic infections or other clinical evidence of immunodeficiency. However, five patients who had splenectomy developed Streptococcus pneumoniae septicemia, usually despite appropriate antibiotic and vaccine prophylaxis. Immunologic Studies The most prominent abnormalities seen with routine immunologic testing of these patients were T-cell and B-cell lymphocytosis, increased numbers of circulating double-negative T lymphocytes, and polyclonal hypergammaglobulinemia. The magnitude of these abnormalities varied (Table). Patients with the most severe lymphoid hyperplasia also had the most pronounced lymphocytosis, the largest numbers of double-negative T lymphocytes, and the highest serum immunoglobulin leve

Sofosbuvir and Ribavirin for Hepatitis C Genotype 1 in Patients With Unfavorable Treatment Characteristics: A Randomized Clinical Trial

IMPORTANCE The efficacy of directly acting antiviral agents in interferon-free regimens for the treatment of chronic hepatitis C infections needs to be evaluated in different populations. OBJECTIVE To determine the efficacy and safety of sofosbuvir with weight-based or low-dose ribavirin among a population with unfavorable treatment characteristics. DESIGN, SETTING, AND PATIENTS Single-center, randomized, 2-part, open-label phase 2 study involving 60 treatment-naive patients with hepatitis C virus (HCV) genotype 1 enrolled at the National Institutes of Health (October 2011-April 2012). INTERVENTIONS In the studys first part, 10 participants with early to moderate liver fibrosis were treated with 400 mg/d of sofosbuvir and weight-based ribavirin for 24 weeks. In the second part, 50 participants with all stages of liver fibrosis were randomized 1:1 to receive 400 mg of sofosbuvir with either weight-based or low-dose 600 mg/d of ribavirin for 24 weeks. MAIN OUTCOMES AND MEASURES The primary study end point was the proportion of participants with undetectable HCV viral load 24 weeks after treatment completion (sustained virologic response of 24 weeks [SVR24]). RESULTS In the first part of the study, 9 participants (90%; 95% CI, 55%-100%) achieved SVR24. In the second part, 7 participants (28%) in the weight-based group and 10 (40%) in the low-dose group relapsed after treatment completion leading to SVR24 rates of 68% (95% CI, 46%-85%) in the weight-based group and 48% (95% CI, 28%-69%; P = .20) in the low-dose group. Twenty individuals participated in a pharmacokinetic-viral kinetic substudy, which demonstrated a slower loss rate of infectious virus in relapsers than in participants who achieved SVR (clearance, 3.57/d vs 5.60/d; P = .009). The most frequent adverse events were headache, anemia, fatigue, and nausea. There were 7 grade 3 events including anemia, neutropenia, nausea, hypophosphatemia, and cholelithiasis or pancreatitis. No one discontinued treatment due to adverse events. CONCLUSION AND RELEVANCE In a population of patients with a high prevalence of unfavorable traditional predictors of treatment response, a 24-week regimen of sofosbuvir and weight-based or low-dose ribavirin resulted in SVR24 rates of 68% and 48%, respectively. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01441180.

Autoimmune Lymphoproliferative Syndrome with Defective Fas: Genotype Influences Penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.

A staged approach to the treatment of Wegener's granulomatosis: Induction of remission with glucocorticoids and daily cyclophosphamide switching to methotrexate for remission maintenance

OBJECTIVE To determine the efficacy of a daily cyclophosphamide (CYC) and glucocorticoid induction and methotrexate (MTX) remission-maintenance regimen for the treatment of Wegeners granulomatosis (WG). METHODS An open-label, prospective, standardized trial for the treatment of WG was performed using CYC and glucocorticoids for remission induction and MTX for remission maintenance. Thirty-one patients were enrolled in this study. Outcome was assessed using predetermined definitions based on clinical characteristics and pathologic, laboratory, and radiographic findings. RESULTS The use of CYC and glucocorticoids for remission induction and MTX for remission maintenance resulted in disease remission for all 31 patients. The median time to remission was 3 months and the median time to discontinuation of glucocorticoids was 8 months. No patients have died, and 5 patients (16%) have had disease relapses at a median of 13 months after achieving remission. Only 2 patients (6%) have had to withdraw from the trial as a result of medication toxicity. CONCLUSION The use of CYC and glucocorticoids for remission induction and MTX for remission maintenance was shown by this study to be an acceptable alternative therapy for patients with active WG, including those with severe disease at onset.

Use of a cyclophosphamide-induction methotrexate-maintenance regimen for the treatment of Wegener's granulomatosis: extended follow-up and rate of relapse.

PURPOSE To determine the relapse rate and outcome in patients with Wegeners granulomatosis treated with daily cyclophosphamide and glucocorticoids to induce remission followed by methotrexate for remission maintenance. METHODS We performed an open-label prospective study in 42 patients with active Wegeners granulomatosis. All patients were treated with a standardized regimen. Outcomes were assessed using predetermined definitions based on clinical characteristics and pathologic, laboratory, and radiographic findings. RESULTS All patients achieved disease remission. The median time to remission was 3 months, and the median time to discontinuation of glucocorticoids was 8 months. During a median of 32 months of follow-up, 1 patient died (of a myocardial infarction not related to vasculitis). Two patients (5%) had to withdraw from the study because of medication toxicity. Twenty-two patients (52%) relapsed, with glomerulonephritis occurring in 16 patients. Of these 16 patients, 4 had an increase of >0.2 mg/dL in serum creatinine level. All 4 patients returned to their prior level of renal function with treatment. None of the 22 relapses met the criteria for severe disease. CONCLUSION The use of cyclophosphamide and glucocorticoids for induction and methotrexate for maintaining remission is an effective and well-tolerated therapeutic approach in patients with active Wegeners granulomatosis.

New Insights into Common Variable Immunodeficiency

Clinical Spectrum of Common Variable Immunodeficiency Dr. Michael C. Sneller (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health [NIH], Bethesda, Maryland): Common variable immunodeficiency (CVI) is a heterogenous syndrome characterized by hypogammaglobulinemia and recurrent bacterial infections. Patients with this syndrome also have an increased incidence of autoimmune disease and malignancy. This rare syndrome has an estimated prevalence ranging from 1:50 000 to 1:200 000 [1, 2]. Unlike X-linked agammaglobulinemia, CVI affects men and women equally. The disorder may occur at any age, but in most patients the onset is in the second or third decade of life [3-5]. This is in contrast to X-linked agammaglobulinemia, in which recurrent infections develop in the first 2 years of life [6]. Infections Clinically, patients with CVI have recurrent bacterial infections of the respiratory tract such as sinusitis, otitis media, bronchitis, and pneumonia. The most common etiologic agents are encapsulated bacteria such as Streptococcus pneumoniae and Haemophilus influenzae. These infections are a direct result of the deficiency in antibody production (specifically immunoglobulin G) that is the hallmark of this syndrome. Immunoglobulin G is the major heat-stable opsonin of extracellular fluid, and the occurrence of repeated infections with encapsulated bacteria in IgG-deficient patients shows the importance of this molecule in host defense against these pathogens. If patients with CVI are not diagnosed (and hence not treated), recurrent pulmonary infections can lead to irreversible chronic lung disease with bronchiectasis. In such patients the spectrum of bacterial pathogens is broader and includes Staphylococcus aureus and Pseudomonas aeruginosa. Septicemia and recurrent infections of the skin, urinary tract, joints, or central nervous system also occur in patients with CVI but are less frequent [3-5]. In rare instances, patients with CVI can become infected with mycobacteria, Pneumocystis carinii, and various fungi. The occurrence of these opportunistic infections may be due to clinically important abnormalities of cell-mediated as well as humoral immunity. Patients with CVI tolerate viral infections normally but some exceptions exist. Herpes zoster (shingles) occurs in up to one fifth of patients with CVI [3]. This represents an abnormally high incidence of viral reactivation in such young patients (ages 20 to 30 years). Frequent and severe recurrences of herpes simplex infections have also been reported in a small number of patients with CVI [7], as have severe cytomegalovirus infections [8]. An unusual syndrome of severe enteroviral infection is seen in patients with primary antibody-deficiency syndromes [9]. This type of infection is most commonly associated with X-linked agammaglobulinemia, but several well-documented cases have occurred in patients with CVI. The most common pathogens are echoviruses, especially echovirus type 11. This syndrome usually occurs as a meningoencephalitis that has a chronic progressive and usually fatal course. Some patients may also have extraneurologic manifestations of disseminated enteroviral infection, including fever, a dermatomyositis-like syndrome, edema, rashes, and hepatitis. In a few cases these manifestations have preceded the development of neurologic symptoms. This syndrome is best diagnosed by examination of the cerebrospinal fluid. Patients almost always have elevated cerebrospinal fluid protein and a lymphocytic pleocytosis; frequently, their cerebrospinal fluid cultures are positive for enterovirus. Gastrointestinal Disease Patients with CVI have various infectious and noninfectious gastrointestinal disorders. The protozoan Giardia lamblia is a common cause of infectious diarrhea in these patients. In normal hosts, Giardia usually causes a self-limited, mild diarrheal syndrome lasting for days to weeks. In contrast, patients with CVI frequently develop chronic diarrhea with clinically significant malabsorption and weight loss that may last for months to years. Diagnosis is made by the presence of cysts or trophozoites in the stool, but because this method is frequently unreliable, diagnosis may require duodenal or jejunal aspirate or duodenal biopsy. Treatment with quinacrine or metronidazole is usually successful, but some patients with CVI require multiple courses of single or combination drug therapy to eradicate Giardia. Patients with CVI are also at increased risk for infection caused by bacterial enteric pathogens such as Salmonella, Shigella, and Campylobacter species. In addition to such infections, we have seen several patients with CVI in whom diarrhea was apparently caused by a fastidious gram-negative bacterium (rod) known as dysgonic fermenter-3 [10]. These patients, who had either acute or chronic diarrhea, negative microbiologic work-up for other enteric pathogens, and dysgonic fermenter-3 isolated from their stool, responded to appropriate antibiotic treatment with resolution of their diarrhea and eradication of this organism from their stool. Thus, in selected patients, stool should be cultured for dysgonic fermenter-3 using cefoperazone-vancomycin agar plates incubated at 35 C [10]. Neoplasms of the gastrointestinal tract (specifically, adenocarcinoma of the stomach and intestinal lymphomas) appear to occur with increased frequency in patients with CVI. In a report from Great Britain [11], 14 of 220 patients with CVI developed malignancies, of which 7 were gastric carcinomas. This represents an incidence of gastric carcinoma 50 times greater than that of the general population. In a U.S. study of 46 patients with CVI, 4 patients had gastric carcinoma [5]. This increased risk should be kept in mind when evaluating upper gastrointestinal symptoms in such patients. Patients with CVI are also at increased risk for developing lymphomas (see below), some of which may primarily involve the bowel. Malabsorption, associated with weight loss and diarrhea, is another gastrointestinal manifestation of CVI. Laboratory findings of malabsorption are usually present and may include hypoalbuminemia, hypocalcemia (due to malabsorption of vitamin D), and decreased levels of vitamin A and carotene. Fecal fat determinations and measurements of d-xylose absorption are usually abnormal. Small-bowel biopsy shows flattening of the villi along with a lymphocytic infiltration in the lamina propria. Histologically, this condition resembles celiac sprue (gluten-sensitive enteropathy) except that plasma cells, a prominent feature of the lymphoid infiltrate in celiac sprue, are absent from the lymphoid infiltrate in patients with CVI. The cause of this syndrome is unknown. Despite the resemblance to celiac disease, gluten-free diets do not correct the malabsorption in most patients, nor has empiric treatment with broad-spectrum antibiotics, corticosteroids, or oral immunoglobulin proven effective. Treatment is supportive with vitamin and mineral replacement as indicated. Inflammatory bowel disease (Crohn disease or ulcerative colitis) appears to occur with increased frequency in patients with CVI [4]; however, before a patient with CVI is diagnosed as having inflammatory bowel disease, a thorough search for an infectious process must be done. Treatment is the same as in nonimmunocompromised patients, except that therapy with immunosuppressive agents should be avoided if possible. Autoimmune Diseases Approximately 20% of patients with CVI will develop one or more autoimmune diseases [3-5], indicating that CVI is a disease of abnormal immune regulation as well as immunodeficiency. Autoimmune (Coomb positive) hemolytic anemia and idiopathic thrombocytopenic purpura are the two most common autoimmune diseases seen [3-5]. Neutropenia is also seen in many patients with CVI, and in some cases antigranulocyte antibodies have been shown [3, 12]. Paradoxically, these patients are unable to mount an antibody response to infecting microorganisms but retain the ability to produce autoantibodies against erythrocytes, platelets, and granulocytes. Treatment of these autoimmune blood dyscrasias in patients with CVI is difficult because standard treatments such as corticosteroids, cytotoxic agents, and splenectomy have the potential to increase further the patients immunodeficiency and susceptibility to infections. High-dose intravenous immunoglobulin has been used successfully in one patient with CVI and autoimmune hemolytic anemia [13] and may be an effective alternative to immunosuppressive therapy in these patients. Pernicious anemia occurs in about 10% of patients with CVI. Although the average age of onset of pernicious anemia in nonimmunodeficient patients is in the sixth decade, patients with CVI usually develop pernicious anemia between the ages of 20 to 40 years [14]. Autoimmune thyroid disease occurs with an increased frequency in patients with CVI. Both Grave disease and hypothyroidism have been reported, with manifestations similar to those in nonimmunodeficient patients. Other autoimmune diseases can occur in association with CVI, including rheumatoid arthritis, systemic lupus erythematosus, and the Sjogren syndrome [3-5]. Lymphoproliferative Disorders Patients with CVI frequently develop lymphoproliferative disorders, which can take several forms. Malignant lymphoma occurs with increased frequency in these patients, although the exact magnitude of this increase is unclear. In the study of 220 patients from Great Britain, 3 lymphomas were seen, which represents a 30-fold increase in the incidence of malignant lymphoma compared with the general population [11]. In a separate U.S. study, 7 lymphomas were seen in 98 patients with CVI [15]. Interestingly, all the lymphomas in this study occurred in female patients. Comparing these data with the age-adjusted figures for the expected incidence of lymphoma in the genera

Pathological Findings in Human Autoimmune Lymphoproliferative Syndrome

The defects in lymphocyte apoptosis that underlie the autoimmune lymphoproliferative syndrome (ALPS) are usually attributable to inherited mutations of the CD95 (Fas) gene. In this report, we present the histopathological and immunophenotypic features seen in the lymph nodes (n = 16), peripheral blood (n = 10), bone marrow (n = 2), spleen (n = 3), and liver (n = 2) from 10 patients with ALPS. Lymph nodes showed marked paracortical hyperplasia. Interfollicular areas were expanded and populated by T cell receptor-alphabeta CD3+ CD4-CD8- (double-negative, DN) T cells that were negative for CD45RO. CD45RA+ T cells were increased in all cases studied. The paracortical infiltrate was a result of both reduced apoptosis and increased proliferation, as measured by in situ detection of DNA fragmentation and staining with MIB-1, respectively. The paracortical proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. Many of the paracortical lymphocytes expressed markers associated with cytotoxicity, such as perforin, TIA-1, and CD57. CD25 was negative. In addition, most lymph nodes exhibited florid follicular hyperplasia, often with focal progressive transformation of germinal centers; in some cases, follicular involution was seen. A polyclonal plasmacytosis also was present. The spleens were markedly enlarged, more than 10 times normal size. There was expansion of both white pulp and red pulp, with increased DN T cells. DN T cells also were observed in liver biopsies exhibiting portal triaditis. In the peripheral blood, the T cells showed increased expression of HLA-DR and CD57 but not CD25. CD45RA+ T cells were increased in the four cases studied. Polyclonal B cell lymphocytosis with expansion of CD5+ B cells was a characteristic finding. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS. Of note, two affected family members of one proband developed lymphoma (T-cell-rich B-cell lymphoma and nodular lymphocyte predominance Hodgkins disease, respectively).

Redistribution, Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and Cytokine Production During First-in-Human Clinical Trial of Recombinant Human Interleukin-15 in Patients With Cancer

PURPOSE Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. PATIENTS AND METHODS We performed a first in-human trial of Escherichia coli-produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. RESULTS Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. CONCLUSION IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration.