Robert C. Hackman

Chronic graft-versus-host syndrome in man : a long-term clinicopathologic study of 20 Seattle patients

This study of chronic graft-versus-host disease (GVHD) describes the clinical, pathologic and laboratory features, and the causes of morbidity and mortality in 20 patients who received allogeneic marrow transplants from HLA identical sibling donors. Chronic GVHD is a pleiotrophic syndrome with variability in the time of onset, organ systems involved and rate of progression. The clinical-pathologic features resemble an overlap of several collagen vascular diseases with frequent involvement of the skin, liver, eyes, mouth, upper respiratory tract, esophagus and less frequent involvement of the serosal surfaces, lower gastrointestinal tract and skeletal muscles. Major causes of morbidity are scleroderma with contractures and ulceration, dry eyes and mouth, pulmonary insufficiency and wasting. Chronic GVHD has features of immune dysregulation with elevated levels of eosinophils, circulating autoantibodies, hypergammaglobulinemia and plasmacytosis of viscera and lymph nodes. In this study, three patients had limited chronic GVHD with relatively favorable prognosis characterized by localized skin involvement and/or hepatic disease without chronic aggressive histology. Most patients, however, had extensive disease with a progressive course. Survival was largely determined by the presence or absence of serious recurrent bacterial infections. The over-all severity of disease was best assessed by using the Karnofsky performance rating.

Adenovirus Infections in Patients Undergoing Bone-Marrow Transplantation

Viral infection is commonly observed after bone-marrow transplantation. We isolated adenovirus from 51 of 1051 patients undergoing marrow transplantation between 1976 and 1982. Of the 46 isolates available for typing, 13 (27.7 per cent) were of the closely related species 11, 34, or 35 (subgenus B). All 13 of the patients with these species had positive urine cultures. The species have previously been associated with the acquired immunodeficiency syndrome or with renal transplantation but are not commonly found in community surveys. Invasive infection was confirmed by biopsy or autopsy in 10 of 51 patients. Seven of the 10 had virus isolated from lung, and 4 died from pneumonia attributed to adenovirus. Two of the five patients with renal isolates had evidence of virally induced renal impairment, and both patients with liver isolates had adenovirus hepatitis. There was no common source that accounted for these adenovirus infections, and the most likely source of infection appeared to be endogenous viral reactivation. The only identifiable risk factor for the development of infection and for severe disease was the presence of moderate to severe graft versus host disease.

Widespread presence of histologically occult cytomegalovirus

Disseminated cytomegalovirus (CMV) has been investigated by in situ hybridization in formalin-fixed paraffin-embedded tissue sections with biotinylated DNA probes. Two cases of disseminated CMV infection were studied at autopsy by this highly specific technique. The presence of CMV in cytomegalic cells is readily shown. In addition, CMV has been detected and localized in many normal-appearing cells. This occult infection occurs in cardiac myocytes, hepatocytes, spleen and lymph node reticular cells, endometrial stromal and glandular cells, and breast stromal cells, as well as in cells in the renal glomerulus, tubule, and interstitium, adrenal cortex and medulla, fallopian tube submucosa, myometrium, and anterior pituitary. Cytomegalovirus infection of endothelial cells has been further documented by immunohistochemical methods utilizing antibody to Factor VIII. These findings suggest that CMV disseminates hematogenously throughout the body, initiating necrotizing foci of infection. The appearance of many diffuse foci suggests that local viral spread occurs via endothelial cell infection. Surprisingly , lymphocyte involvement was not observed.

Human Herpesvirus 6 in Lung Tissue from Patients with Pneumonitis after Bone Marrow Transplantation

BACKGROUND Human herpesvirus 6 (HHV-6) is a recently described herpesvirus that is epidemiologically and biologically similar to cytomegalovirus. It is the cause of exanthem subitum (roseola) in children. METHODS To evaluate the possible role of HHV-6 infection in pneumonitis in immunocompromised patients, we used quantitative HHV-6 polymerase chain reactions to study lung-biopsy specimens from 15 patients with pneumonitis after bone marrow transplantation and lung tissue from 15 immunocompetent subjects without pneumonitis and 6 fetuses. RESULTS HHV-6 DNA was detected in lung tissue from all 15 patients, from 14 seropositive control subjects, and from none of the 7 seronegative control subjects. Six patients had levels of HHV-6 DNA in lung tissue that were 10 to 500 times higher than those in any of the other patients or control subjects. Increased levels of HHV-6 DNA correlated with a decreased risk of death from pneumonitis (P = 0.015), an increased severity of graft-versus-host disease (P = 0.023), and the presence of idiopathic pneumonitis (P = 0.037). Levels of HHV-6 DNA correlated directly with the changes in HHV-6 antibody titers in the interval between the pretransplantation period and the open-lung biopsy (P = 0.002). Low levels of HHV-6 antibody at the time of the open-lung biopsy were also associated with the diagnosis of idiopathic pneumonitis (P = 0.002). CONCLUSIONS The concentrations of HHV-6 genome in lung tissue and their relation to changes in serologic titers support an association between HHV-6 infection and idiopathic pneumonitis in immunocompromised hosts.

Herpes Simplex Virus Pneumonia: Clinical, Virologic, and Pathologic Features in 20 Patients

Herpes simplex virus (herpesvirus) was isolated from autopsy lung specimens of 20 patients with clinical, roentgenographic, and histologic evidence of pneumonia. Mucocutaneous herpesvirus infection preceded the onset of pneumonia in 17. Twelve patients had focal pneumonia, 10 of whom had concomitant herpetic tracheitis, esophagitis, or both. Eight patients had diffuse interstitial pneumonia, six of whom had dissemination of herpesvirus to the other organs. Of the eight lung isolates available for typing, seven were herpesvirus-1 and one, herpesvirus-2. A high prevalence of herpesvirus antibody in serum samples obtained before pneumonia and identical restriction endonuclease patterns between mucosal and lung isolates in individual patients indicated that, in most cases, herpesvirus pneumonia was due to endogenous reactivation of virus. Focal herpesvirus pneumonia appeared to result from contiguous spread of herpesvirus to lung parenchyma, whereas diffuse interstitial pneumonia appeared to be a manifestation of hematogenous dissemination of virus.

Etiology and outcome of diarrhea after marrow transplantation: A prospective study

BACKGROUND/AIMS Acute diarrhea after marrow transplant is usually ascribed to acute graft-vs.-host disease (GVHD) or infection, with a reported 40%-50% incidence of infection. The aim of this study was to determine the incidence of acute diarrhea after transplantation, its causes, and its outcome. METHODS Two hundred ninety-six patients were followed up; patients with diarrhea were studied using standard evaluation of stool plus immunoelectron microscopy; assays for astrovirus, picobirnavirus, and Norwalk virus; and gene-probe methods for toxin-producing Escherichia coli. In 38 patients with diarrhea, intestinal biopsy specimens and duodenal fluid were also analyzed. RESULTS One hundred fifty acute diarrheal episodes developed in 126 patients (an incidence of 43%). Intestinal infection was found in 20 of 150 episodes: viruses (astrovirus, adenovirus, cytomegalovirus, and rotavirus) in 12 patients, nosocomially acquired bacteria (Clostridium difficile and Aeromonas) in 7 patients, and mixed infection in 1 patient. Acute GVHD was responsible for 72 of 150 episodes (48%). Clinical signs and symptoms of infection and GVHD were similar. In 58 of 150 episodes (39%), no clear etiology could be found for self-limited diarrhea. CONCLUSIONS Intestinal infection accounted for 13% and acute GVHD for 48% of diarrheal episodes. The most common infecting organisms were astrovirus, C. difficile, and adenovirus. Most cases of diarrhea after marrow transplant are not caused by infection.

Brief Communication: Fatal Human Metapneumovirus Infection in Stem-Cell Transplant Recipients

Context Human metapneumovirus (hMPV) is a newly discovered respiratory virus. Contribution This study from a cancer referral center retrospectively examined bronchoalveolar lavage samples collected from 1995 to 1999 from hematopoietic cell transplant recipients with new or changing pulmonary infiltrates. The authors cultured hMPV in 5 of 163 symptomatic patients who were thought to have had the idiopathic pneumonia syndrome. Clinical findings included fever, cough, nasal congestion, respiratory failure, pulmonary hemorrhage, and culture-negative sepsis. Four of the patients died. Cautions All patients had moderate to severe symptoms. Implications Human metapneumovirus may cause respiratory failure in immunocompromised adults. The Editors Human metapneumovirus (hMPV) is a newly discovered respiratory virus associated with 10% to 25% of respiratory tract infections in young children (1, 2). This virus has been detected in respiratory disease at all ages (3, 4), although lower respiratory tract disease occurs more frequently in very young and very old persons and in those with underlying conditions. Clinical findings seen with hMPV infection are similar to those of respiratory syncytial virus (RSV) infection (5). As with RSV, serious disease associated with hMPV infection has been reported in immunocompromised patients. Deaths have been reported in a child (5), adults with hematologic malignant conditions (6), and adults after bone marrow or organ transplantation (7, 8). Pneumonia remains one of the most frequent serious complications of hematopoietic stem-cell transplantation (HCT) (9). Despite increased testing for pathogens in the transplantation setting, nearly 10% of myeloablative transplant recipients still develop the idiopathic pneumonia syndrome (10). We retrospectively evaluated archived bronchoalveolar lavage (BAL) fluid from patients undergoing HCT by using a sensitive hMPV-specific, real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay to determine whether hMPV may be a cause of pulmonary morbidity and mortality in this setting. Methods Patients undergoing HCT who underwent BAL between 1995 to 1999 for evaluation of respiratory disease were eligible for inclusion if they had previously consented to diagnostic studies on their tissue and if residual specimens were available and were in good condition. We retrospectively reviewed clinical and laboratory records, biopsy specimens, and autopsy specimens of all patients with detected hMPV. We also evaluated stored BAL samples in 19 HCT recipients who were enrolled in a separate prospective study evaluating BAL after transplantation. Specimens Our institution has used a diagnostic algorithm for defining the cause of pneumonia for patients with new or changing pulmonary infiltrates since 1994 (11). We processed BAL samples in a centralized laboratory for pathogens, as previously described (11). Biopsy and autopsy specimens were submitted for routine bacterial, fungal, and acid-fast bacilli culture tests; underwent cytologic examination; and were tested for mycobacteria, Legionella species, Pneumocystis jiroveci, and fungal species. Additional studies performed in real time included culture and antibody staining of BAL fluid for cytomegalovirus (CMV), RSV, parainfluenza, and influenza viruses. For our study, we assayed BAL from patients with hMPV by using PCR for RSV (12), hMPV (13), parainfluenza types 1 and 3, influenza A and B, adenovirus, human herpesvirus-6 (14), and Aspergillus species (15). In addition, we performed broad-range fungal 18S rDNA PCRs to detect other fungal pathogens (16) and a -globin PCR (17) to determine whether amplifiable DNA was present in BAL fluid. The RT-PCR assay used probes that were designed to amplify 69 and 74 base-pair fragments of the fusion protein genes from the 2 major hMPV groups (13). This assay was sensitive to 10 copies per reaction and was specific for hMPV, as previously described (18). We also examined lung tissue from autopsy specimens of patients with known RSV, CMV, and hMPV infection near the time of death after tissue digestion by using the same RT-PCR methods. Paraffin-embedded tissues were examined for the presence of hMPV antigen by immunohistochemistry in the laboratory of Dr. Thijs Kuiken, Erasmus University, Rotterdam, the Netherlands (19). We included lung sections of an experimentally infected cynomolgus monkey with hMPV as positive controls. Role of the Funding Sources The National Institutes of Health, the Fred Hutchinson Cancer Research Center, and the Adult Leukemia Research Center provided funding for this study. The funding sources had no role in the design, analysis, interpretation, or writing of this manuscript or in the decision to submit the manuscript for publication. Results Patients To determine the presence of hMPV, we evaluated BAL specimens obtained throughout the calendar year from 163 symptomatic and 19 asymptomatic HCT recipients. The mean age of patients undergoing BAL was 42 years; only 10 patients (7%) were younger than 20 years of age. Most patients (99 of 163 patients [60%]) with BAL specimens had received allogenic bone marrow transplants from unrelated donors (57 of 163 patients [35%]), matchedrelated donors (24 of 163 patients [15%]), or mismatchedrelated donors (15 of 163 patients [9%]). Another 64 patients received peripheral blood stem-cell transplants (64 of 163 patients [39%]) from matched allogenic donors (16%), autologous donors (16%), or unrelated donors (4%). One patient received an unrelated cord blood stem-cell transplant. We detected hMPV in the tested BAL specimens of 5 of 163 patients (3.0%) and in 8 of 211 specimens (3.8%). Patients were between 24 and 54 years of age, and they had received different preparative regimens and sources of stem cells (Table). We detected hMPV in samples obtained between January and April. Clinical signs and symptoms around disease onset included a low-grade fever in 4 patients, cough in 3 patients, nasal congestion in 3 patients, and sore throat in 4 patients (although transplantation-related mucositis may have confounded some symptoms). Three patients had wheezing at diagnosis. We did not note sinusitis, otalgia, otitis media, conjunctivitis, or skin rashes. All patients eventually developed radiographic evidence of diffuse pulmonary disease, and we noted rapid progression of pulmonary infiltrates in 4 of 5 patients (Figure, top). Table. Characteristics of Hematopoietic Stem-Cell Transplant Recipients with Human Metapneumovirus Infection Figure. Computed tomography scan of chest in a patient with fatal human metapneumovirus infection after transplantation ( top ) and lung histologic study in a patient with human metapneumovirus pneumonia ( bottom ). Top. Bottom. inset arrow arrow All 5 cases of hMPV lower respiratory tract disease occurred early (<40 days) after transplantation. Lymphopenia was common, although 3 patients had neutrophil engraftment at the time of hMPV detection. Diffuse alveolar hemorrhage was present in 3 patients, and prominent alveolar macrophages were present in another patient. The quantity of hMPV ranged from 2104 RNA copies/mL to 3108 RNA copies/mL of BAL fluid (Table). Detection of Potential Co-Pathogens All BAL sample test results for patients with hMPV were negative for CMV, RSV, influenza viruses, adenovirus, and fungi by real-time PCR. We did not identify any accompanying pathologic or microbiological finding in 3 patients. Herpes simplex virus was isolated at autopsy in case 3, and parainfluenza was detected by culture and PCR in case 4. Case 2 had CMV isolated from lung tissue at autopsy only. We also tested BAL samples from patients with hMPV for human herpesvirus-6. Four of eight sample test results were negative, and the remaining 3 samples had low copies of human herpesvirus-6 DNA (53, 71, and 93 human herpesvirus-6 DNA copies/mL), which are levels compatible with detection of virus in latently infected cells (14). Treatment and Outcome We treated 3 of 5 patients who received a diagnosis of diffuse alveolar hemorrhage (cases 1, 4, and 5) with high-dose corticosteroids without discernable benefit. The hMPV viral load in case 4 decreased despite the use of corticosteroids (Table). Case 2 received aerosolized ribavirin for parainfluenza pneumonia, but additional respiratory samples were unavailable for testing. Progression of respiratory symptoms was rapid, with a median of 4 days (range, 1 day to 32 days) between initiation of oxygen therapy and death. Four patients died of respiratory complications at a median of 16 days (range, 1 day to 35 days) after the BAL was found to be positive for hMPV. Three patients (cases 1, 2, and 3) also had a culture-negative sepsis syndrome that required pressor agents. Clinical diagnoses at the time of death included idiopathic pneumonia in 3 patients; sepsis, shock, or both in 3 patients; and parainfluenza pneumonia in 1 patient. The mortality rate within 3 months of the first BAL in patients with hMPV was significantly higher than that in patients in whom hMPV was not detected (Wilcoxon rank-sum test; P= 0.014). We performed a limited postmortem examination of the lungs for case 2. We noted several bilateral pleural adhesions with diffuse alveolar damage and moderate hemorrhage. The cytologic changes suggested viral infection (Figure, bottom). Postmortem lung culture grew CMV that had not been previously detected. One of 2 archived frozen tissue samples, obtained at autopsy 8 days after bronchoscopy, was found to be positive for hMPV by RT-PCR but was negative by immunohistochemistry. One HCT recipient survived hMPV infection early in her transplantation course. This young patient had neutrophil engraftment when respiratory symptoms were noted. Nonetheless, she experienced a rapidly deteriorating respiratory course and, although she was never intubated, required supplemental oxygen for 2 months. Her hoarseness, dyspnea, and cou

The effect of prophylactic fluconazole on the clinical spectrum of fungal diseases in bone marrow transplant recipients with special attention to hepatic candidiasis. An autopsy study of 355 patients.

We reviewed 355 autopsies performed between 1990 and 1994 at a major marrow transplant center to determine whether fluconazole prophylaxis prevented visceral fungal infection. Fluconazole prophylaxis was defined by a minimum of 5 prophylactic doses. Fungal infection (any site) was found in 40% of patients transplanted and autopsied at the center. Overall, the proportion of autopsies with any fungal infection was not different for those patients receiving no fluconazole prophylaxis versus those with prophylactic fluconazole. With fluconazole prophylaxis, candidal infections were less frequent, decreasing from 27% to 8%, while Aspergillus infections were more frequent, increasing from 18% to 29%. No increase in deaths related to non-albicans Candida infections was seen. Of the 329 patients with livers examined, hepatic infection caused by Candida species was significantly less common in patients who had received fluconazole. Fungal liver infection was found in 31 patients (9%), 16% of those who were not treated with fluconazole and 3% of those who were treated with fluconazole. Since patients with candidal infections died earlier after marrow transplant than patients with mold infections, we speculate that a longer length of survival may dispose toward acquisition of mold infections. Fluconazole prophylaxis in this cohort of marrow transplant patients undergoing autopsy resulted in a significant reduction in infection caused by Candida species and an increase in mold infections.

Safety and immunologic effects of IL-15 administration in nonhuman primates

The administration of cytokines that modulate endogenous or transferred T-cell immunity could improve current approaches to clinical immunotherapy. Interleukin-2 (IL-2) is used most commonly for this purpose, but causes systemic toxicity and preferentially drives the expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cells, which can inhibit antitumor immunity. IL-15 belongs to the gamma(c) cytokine family and possesses similar properties to IL-2, including the ability to induce T-cell proliferation. Whereas IL-2 promotes apoptosis and limits the survival of CD8(+) memory T cells, IL-15 is required for the establishment and maintenance of CD8(+) T-cell memory. However, limited data are available to guide the clinical use of IL-15. Here, we demonstrate in nonhuman primates that IL-15 administration expands memory CD8(+) and CD4(+) T cells, and natural killer (NK) cells in the peripheral blood, with minimal increases in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Daily administration of IL-15 resulted in persistently elevated plasma IL-15 levels and transient toxicity. Intermittent administration of IL-15 allowed clearance of IL-15 between doses and was safe for more than 3 weeks. These findings demonstrate that IL-15 has profound immunomodulatory properties distinct from those described for IL-2, and suggest that intermittent administration of IL-15 should be considered in clinical studies.

Rapid Detection of Cytomegalovirus Pulmonary Infection by Bronchoalveolar Lavage and Centrifugation Culture

Cytomegalovirus infection remains a major cause of morbidity and mortality in marrow transplant recipients. Results of a rapid centrifugation viral culture of bronchoalveolar lavage specimens from 33 marrow transplant recipients with pneumonia were compared with those for conventional viral culture of concurrently or subsequently obtained lung tissue. The centrifugation culture results were also compared to results of cytologic and immunochemical examination of these specimens. Centrifugation culture was positive within 16 hours of inoculation in 22 of 23 (96%) specimens from patients with positive conventional culture of lung tissue. Detection of cells positive for cytomegalovirus by immunofluorescent antibody staining or cytologic identification was less sensitive (59% and 29%, respectively). There was no evidence of cytomegalovirus in specimens from patients without evidence of cytomegalovirus pulmonary infection by any technique. The sensitivity (96%) and specificity (100%) of centrifugation culture of specimens from marrow transplant recipients approach that of viral culture of lung tissue.