Gerhard R. F. Krueger

Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates

Fifteen human herpesvirus-6 (HHV-6) isolates from normal donors and patients with AIDS, systemic lupus erythematosis, chronic fatigue syndrome, collagen-vascular disease, leukopenia, bone marrow transplants, Exanthem subitum (roseola), and atypical polyclonal lymphoproliferation were studied for their tropism to fresh human cord blood mononuclear cells, growth in continuous T cell lines, reactivity to monoclonal antibodies, and by restriction enzyme banding patterns. All isolates replicated efficiently in human cord blood mononuclear cells, but mitogen stimulation of the cells prior to infection was required. The ability to infect continuous T-cell lines varied with the isolates. Isolates similar to GS prototype infected HSB2 and Sup T1 cells and did not infect Molt-3 cells, whereas isolates similar to Z-29 infected Molt-3 cells but not HSB2 and Sup T1 cells. Some of the monoclonal antibodies directed against the HHV-6 (GS) isolate showed reactivity with all isolates tested, but others only reacted with HHV-6 isolates similar to the GS isolate and not with those similar to Z-29 isolate. Restriction enzyme analysis using EcoRI, BamHI, and HindIII revealed that HHV-6 isolates from roseola, bone marrow transplant, leukopenia, and an HIV-1-positive AIDS patient from Zaire (Z-29) were closely related but distinct from GS type HHV-6 isolates. Based on the above findings, we propose that, like herpes simplex virus types 1 and 2, the 15 HHV-6 isolates analyzed can be divided into group A (GS type) and group B (Z-29 type).

Human B-lymphotropic virus (human herpesvirus-6).

Human B-lymphotropic virus (HBLV), also known as human herpesvirus-6 (HHV-6) was first isolated in 1986 from AIDS patients and patients with other lymphoproliferative disorders. HBLV is distinct from known human herpesviruses, biologically, immunologically and by molecular analysis. HBLV can infect and replicate in fresh and established lines of hemopoietic cells and cells of neural origin, suggesting wide tropism. The prevalence of HBLV antibody in the normal population was 26% though clear differences between different populations were observed. The prevalence of HBLV antibody an elevated antibody titer was higher in sera from certain malignancies, Sjögrens syndrome and sarcoidosis. Antibody to HBLV was also elevated in AIDS patients and patients with chronic fatigue syndrome. HBLV-DNA was detected in some B-cell lymphomas. The broad in vitro tropism, combined with immunological and molecular evidence of HBLV infection in individuals raise the question of the pathogenicity of this virus in some diseases. Because in vitro co-infection of CD4 cells by HBLV and HIV leads to enhanced degeneration, this raises the possibility that infection in AIDS patients by both viruses can aggravate the HIV-induced immunodeficiency. Specific reagents and immunological and molecular assays are currently being investigated, which will aid in virus detection in cells from patients, and in elucidating the possible pathogenesis of HBLV.

Polymerase chain reaction amplification and in situ hybridization for the detection of human B-lymphotropic virus

Polymerase chain reaction amplification (PCR) is a recently described technique that allows for the amplification of a given sequence of DNA. It can be used to reliably amplify sequences of up to 3 kb within hours. The amplified sequence can then be recognized by hybridization with a specific probe after transfer onto nitrocellulose or nylon paper. We used PCR to recognize human B-lymphotropic virus (HBLV or HHV-6) specific sequences in various tumors as well as in the blood of patients with AIDS. Sixty-three specimens of DNA extracted from peripheral blood of patients with AIDS as well as DNA extracted from various lymphoproliferative disorders were analysed; 52 out of 63 (83%) patients with AIDS were found to have amplification of the HHV-6 specific sequence; 2 out of the 63 (3%) had equivocal amplification and 9 (14%) were found to be negative. Twenty out of 23 tumors were found to have amplified HBLV-specific sequences. Only one of these tumors was positive by Southern hybridization on restriction enzyme digested genomic DNA. In situ hybridization of clinical specimens using radiolabelled RNA probes or hapten-labelled DNA probes was used to detect the presence of HBLV in tumors. Three tumors of B cell origin were found to be positive for HBLV.

p53 and Ki 67 expression in preneoplastic and neoplastic lesions of the oral mucosa

Some lesions of the oral mucosa such as leukoplakia and erythroplakia may develop into squamous cell carcinoma (SCC). At present, however, there is no method available to predict malignant transformation. It is known that the grade of dysplasia is related to the potential malignant development, but this is unreliable as the only indicator. In 64 hyperplastic lesions and 85 SCC of the oral mucosa, a correlation between the expression of the mutated tumor-suppressor gene p53 and the dysplasia of the lesions was found. Ki 67 was used as a proliferation marker. The results imply that expression of mutated p53 is an indicator for potential malignant development in benign lesions of the oral mucosa.

Antibody prevalence to HBLV (human herpesvirus-6, HHV-6) and suggestive pathogenicity in the general population and in patients with immune deficiency syndromes

Detailed serologic screening showed an antibody prevalence to HBLV (HHV-6) in the general population of 26% if very strict criteria for antibody positivity were applied. Lower and borderline antibody titers yet may be found in up to 63% of the population. Only 17% of these persons have clinical symptoms; in the majority infection remains silent. HHV-6 infection apparently occurs already quite early in life, and initial symptoms can occur, such as short-term high fever, sore throat, local lymphadenopathy and skin rash. Lesions disappear without specific treatment. The frequency of positive antibody tests at higher titers rises in patients with immune deficiency and with atypical lymphoproliferative diseases to 60 and 75%. The rise in antibody titers is associated in patients with immune deficiency by characteristic shifts of blood lymphocyte populations, essentially by increase in immature T-lymphocytes. Highest titers are found in patients with lymphoproliferative syndromes, yet the percentage of atypical lymphoid cells harboring the viral genome is low (about 2% of seropositive patients). Thus it appears, that HBLV, similar to other herpesviruses such as Epstein-Barr virus, usually causes a silent seroconversion, yet may be associated with variable clinical pathology when persisting in an active state. Its pathogenic effect might be rather a cofactor contributing to immune disturbance than overt oncogenicity.

The Impact of Declining Clinical Autopsy: Need for Revised Healthcare Policy

In Western countries, autopsy rates for patients deceased in hospitals have dropped to record lows, while the average frequency of major errors in clinical diagnoses has more than doubled during the same time period. Meanwhile, the Institute of Medicine and the U.S. Department of Health and Human Services have called attention to the high frequency of errors affecting patient safety, bringing the issue of public safety to the forefront of public health concerns. Although autopsies represent a vital tool for the acquisition of new medical knowledge and for medical quality assurance, health care professionals, insurers, and politicians apparently have not chosen the right approach to solve the problem of declining autopsy rates. The present article reviews the current status of clinical autopsies and addresses causes and consequences of their neglect and appeal the urgent need to revise the policy for clinical autopsy.

p53 and PCNA expression in carcinogenesis of the oropharyngeal mucosa

Hyperplastic lesions of the oral mucosa such as leukoplakia and oral lichen planus can eventually develop into squamous cell carcinomas (SCC) and provide an excellent model for multistage carcinogenesis. The development of carcinomas is assumed to be the result of interaction of genetic factors, locally applied carcinogens and immunological unresponsiveness. The purpose of this study was, therefore, to determine the role of alterations of the tumour suppressor gene p53, and the proliferation status of the lesions determined by PCNA expression. We investigated p53 and PCNA expression in 265 tissue sections of normal mucosa, premalignant, malignant and metastatic lesions of the oral mucosa by immunohistology. Quantitative analysis showed a gradual increase in PCNA expression from normal mucosa to moderately differentiated SCC. p53 expression was detectable in benign premalignant lesions. The increase in the number of p53-positive biopsies was correlated with the dysplasia and loss of differentiation in the premalignant and malignant lesions.

pH-stabilization of predegraded PDLLA by an admixture of water-soluble sodiumhydrogenphosphate--results of an in vitro- and in vivo-study.

Aim of the study was to examine if the addition of buffering sodiumhydrogenphosphate to poly(D,L)lactide(PDLLA) would stabilize the pH-value in the in vivo environment of implanted material and whether this improves its biocompatibility. The material was predegraded just to the point of viscous disintegration to test the PDLLA in the moment of its most aggressive effect on the surrounding tissue. Racemic amorphous PDLLA was injection-molded with or without the admixture of 1 mol NaP per 100 mol lactate, the degradation product of PDLLA (=1 mol%) to form 20mm x 3 mm x 2mm rods. Predegradation was performed by storing the rods at 55 degrees C for 14 days, just to the point of beginning dissolution. Predegraded PDLLA or PDLLA + NaP samples were used for in vitro incubation tests, as well as for the in vivo study, where the rods were implanted into the spinal muscles of 30 male Wistar rats. Repeatedly, measurements of the pH-value were made in the incubation solutions in vitro. The surrounding tissue of the implanted samples as well as the normal contralateral muscle tissue was checked for its pH-value in a group of 3 rats, respectively, anaesthesized at various time intervals after implantation. After these measurements the implants and their surrounding tissues were excised for histological examination. In Ringers solution pH-values dropped immediately within the first week of incubation of both predegraded materials reaching -4 pH units after 4 weeks in the PDLLA containing medium, after 6 weeks in the PDLLA + NaP containing medium. Soerensen buffer slowed the pH decrease with significant differences between the material groups up to the 28th week. In vivo, the pH of the surrounding tissue was influenced by the implanted PDLLA material up to the 4th week, while the admixture of NaP resulted in a significant pH stabilization. A higher quantity of macrophages and giant cells were seen between the 2nd and 6th week after the implantation in the environment of pure PDLLA compared with PDLLA + NaP. Complete resorption of predegraded pure PDLLA or PDLLA + NaP from the extracellular space was reached 28 weeks postimplantation in vivo. Thus, sodiumhydrogenphosphate improves the biocompatibility of degrading PDLLA at the point of viscous disintegration by stabilizing the pH-value in the environment of the implants for several weeks and reducing adverse tissue reactions.

Cysts associated with longstanding impacted third molars

Three patients are described in whom large cysts developed around third molars that had purposely been left in place. The cases presented emphasize the need for an adequate prospective study to evaluate the long-term morbidity of asymptomatic third molars that are left in place.

Diagnosis and differential diagnosis of progressive lymphoproliferation and malignant lymphoma in persistent active herpesvirus infection

Persistent active Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6) infections may be accompanied by extensive lymphoproliferative reactions mimicking malignant lymphoma. Such atypical polyclonal lymphoproliferations (APL) can frequently be separated from malignant lymphoma only by detailed immunopathologic and virologic studies. Recommended procedures include immunotyping of lymphoid cells, extensive viral serology, in situ hybridization and Southern blotting for viral DNA, gene rearrangement and DNA ploidy studies. Clear separation of APL from malignant lymphoma is essential for therapeutic planning.