Helmuth Schmidt

Polymerase chain reaction to evaluate antiviral therapy for cytomegalovirus disease

A sensitive and specific method to monitor suppression of cytomegalovirus (CMV) replication is essential in patients treated with ganciclovir after allogeneic bone-marrow transplantation. In this study, antiviral therapy of eighteen episodes of symptomatic CMV infection in 15 such patients were followed up clinically and by virus culture and polymerase chain reaction (PCR). Clinical improvement, culture, and PCR were assessed for their ability to predict the efficacy of ganciclovir therapy in each patient. In eleven successfully treated episodes of CMV disease (disappearance of symptoms and improvement in biochemical variables) clinical improvement was associated with an effective suppression of virus replication as shown by negative culture and PCR assays of blood and urine specimens obtained after antiviral therapy. 1 patient who did not improve clinically when receiving antiviral therapy remained both culture positive and PCR positive for CMV. 6 patients with early relapse of CMV disease or who died after an initial clinical improvement were PCR positive but culture negative after termination of therapy. Demonstration of CMV in blood and urine by PCR after stopping antiviral therapy (even when culture is negative) points to incomplete suppression of virus replication. The findings show that PCR is a better predictor of the efficacy of antiviral therapy than are culture or clinical assessment.

Genetic and serological heterogeneity of the supertypic HLA-B locus specificities Bw4 and Bw6

Gene cloning and sequencing of theHLA-B locus split antigens B38 (B16.1) and B39 (B16.2) allowed localization of their subtypic as well as their public specificities HLA-Bw4 or-Bw6 to the α-helical region of the α 1 domain flanked by the amino acid positions 74–83. Comparison of their amino acid sequences with those of otherHLA-B-locus alleles established HLA-Bw6 to be distinguished by Ser at residue 77 and Asn at residue 80. In contrast, HLA-Bw4 is characterized by at least seven different patterns of amino acid exchanges at positions 77 and 80–83. Reactivity patterns of Bw4-or Bw6-specific monoclonal antibodies reveal two alloantigenic epitopes contributing to the HLA-Bw4 or-Bw6 specificity residing next to the region of highest diversity of the α 1 domain.

Membranous nephropathy after bone marrow transplantation in ciclosporin treatment.

Dr. G.A. Müller, Medizinische Klinik, Abteilung für Innere Medizin III, Otfried-Müller-Strasse, D-7400 Tübingen 1 (FRG) Dear Sir, We report here the occurrence of membranous nephropathy (MN) in a 20-year-old patient with severe aplastic anaemia during treatment with cyclosporin (CS-A) after bone marrow grafting from his HLA-identical brother. Conditioning therapy was performed on days -5 to -2 before bone marrow transplantation (BMT) using cyclophosphamide 50 mg/b.w./day. To prevent graftversus-host disease (GvHD), CS-A was given from day -1 on as usual [1]. As infectious disease prophylaxis he received trimethoprime and sulfamethoxazole, as well as anti-CMV hyperimmunoglobulin, acyclovir and ampho-tericin B, as described in detail earlier [1]. Four weeks after BMT, a trilinear take was documented. Skin GvHD grade I appeared 10 days after BMT and was successfully treated with prednisolone 1 mg/b.w./day. Seven months after BMT, proteinuria was detected for the first time and the patient developed a nephrotic syndrome (maximal proteinuria of 30 g/day). Histologi-cal, immunohistological as well as electron microscopical analysis of a renal biopsy revealed MN stage I [2]. Furthermore, signs of CS-A toxicity in proximal tubular epithelial cells were observed. In addition, interstitial cellular infiltrates characterized by specific monoclonal 1 Supported by the Deutsche Forschungsgemeinschaft DFG, Mu 523/3–2 and SFB 120. antibodies were shown to consist mainly of CD4 + /CD8 + Tlymphocytes with aCD8 + /CD4+ ratio of 1.9, as well as of a large number of monocytes/macrophages. Expression of different MHC class II products on renal epithelial cells and on vessels did not differ from those of the normal kidney. At the time of renal biopsy, cholestatic enzymes were elevated; anti-nuclear and anti-IgM antibodies against rubella, but no anti-DNA antibodies were found in the patient’s serum. Since CS-A toxicity had been documented by renal biopsy, CS-A therapy was stopped and MN was treated with chlorambucil and prednisolone [3]. During three therapeutical cycles, proteinuria declined rapidly to 2.5–5.0 g/day. At the end of the third cycle the patient presented with a relapse of proteinuria (9.0 g/day). The therapeutic regimen was changed to CS-A and low-

Relative Roles of Natural Killer- and T Cell-Mediated Anti-Leukemia Effects in Chronic Myelogenous Leukemia Patients Treated with Interferon-α

Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.

Desensitization of platelets to iloprost. Loss of specific binding sites and heterologous desensitization of adenylate cyclase.

The binding characteristics of [3H]prostacyclin and [3H]iloprost ([3H]5-[(E)-(1S,5S,6R,7R)-7-hydroxy-6-[(E)-(3S,4RS) -3-hydroxy-4-methyl-1-octen-6-inyl]-bicyclo[3.3.0]octan-3-yl idene] -pentanoic acid) and platelet adenylate cyclase activities were investigated in platelet-rich plasma preincubated with iloprost. The exposure of platelets to 0.1 microM iloprost (12 h, 20 degrees C) caused a significant loss of iloprost binding sites (P less than 0.01) without causing changes in binding affinity. This loss of specific [3H]iloprost binding was time- and dose-dependent. The reduction of iloprost receptor density was accompanied by an impaired responsiveness of platelet adenylate cyclase to iloprost, prostaglandin D2 and forskolin. In contrast, basal adenylate cyclase activity was not affected by iloprost pretreatment. The diminished response of the enzyme to GTP and NaF pointed to an involvement of the stimulatory guanyl nucleotide-binding protein (Gs) in iloprost-induced heterologous desensitization. Consequently, [32P]NAD+ and cholera toxin were used for the direct labelling of Gs. Platelet membranes desensitized to iloprost incorporated less label into the 45 kD subunit of Gs. These data suggest that the site of action of iloprost for heterologous desensitization of human platelet adenylate cyclase is located on Gs.

Differential regulation of HLA class I genes by interferon

Allele-specific differences in the regulation of HLA class I genes by type I interferon (IFN) were observed after transfection of eight HLA-B,-A, or-C genes into mouse L cells. HLA-B7 and -Bw64 gene expression was significantly more inductible by type I IFN than the genes coding for HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3, and HLA-A2 antigens. Modification of the 5′ end of HLA-B7 and HLA-B27 genes revealed the presence of enhancer sequences responding to IFN treatment in the 5′ untranslated region of HLA-B7, but not of HLA-B27 and suggested further, independently acting enhancer elements downstream of the transciption initiation site. Comparison of 5′ enhancer region sequences in correlation with type I IFN inducibility of the different HLA class I alleles indicated that the exchange of only two nucleotides in the interferon response sequence (IRS) or enhancer A region of HLA-B7 or -Bw64 could account for nonregulated promoters in all other HLA-A,-B or -C alleles analyzed. Thus, type I IFN stimulation of HLA class I genes in mouse L cells appears to predominantly operate in most alleles by a mechanism targeted to enhancer sequences downstream of the genes transcription initiation site.

Philadelphia-positive chronic myelogenous leukemia with breakpoint 5′ of the breakpoint cluster region but within the bcr gene

SummaryWe report on the first Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) characterized by a rearrangement within the 5′ part of the bcr gene on chromosome 22, but outside the restricted breakpoint cluster region. In situ hybridization studies revealed a translocation of the c-abl oncogene to the Ph chromosome and Northern blot analysis identified a chimeric 8 kb bcr/abl RNA transcript in leukemic cells. These data suggest that 1. less bcr coding sequences than previously assumed may be essential for the putative transforming activity of the rearranged bcr/abl gene and 2. the bcr probes currently used for diagnostic purposes could miss Ph-positive CML cases.

Cytomegalovirus in liver biopsies of marrow transplant recipients: detection methods, clinical, histological and immunohistological features

In a retrospective analysis liver biopsy specimens obtained from 44 marrow transplant recipients were studied to evaluate the frequency of local presence of human cytomegalovirus (CMV) and graft-versus-host disease (GvHD)-like histological and immunohistological alterations in patients with and without liver dysfunction following bone marrow transplantation (BMT). In 22 of 28 patients with marked liver dysfunction after BMT and histopathological alterations described as typical for acute GvHD CMV could be detected in the liver biopsy specimen. The polymerase chain reaction (PCR) technique revealed the highest sensitivity for CMV detection in liver biopsy samples, but in 20 of 22 PCR-positive specimens CMV infection could be confirmed by at least one additional technique. All the liver biopsies obtained from 16 patients with normal liver function lacking histopathological signs of GvHD were CMV negative. In all 3 patients with CMV-positive liver biopsy started on antiviral therapy liver function improved and no generalized CMV disease occurred. All the 4 patients without local presence of CMV started on severe immunosuppressive therapy showed an improvement of liver dysfunction without occurrence of CMV infection. Local CMV infection of the liver could not be differentiated from hepatic GvHD by clinical and histopathological features, nor by immunohistological analysis of the bile duct epithelium. In contrast, only in liver biopsy with local viral presence could an increase in HLA class II- and ICAM-1 expression be demonstrated on hepatocytes. Thus, especially the high negative predictive value of the PCR technique helps to manage the patient with liver dysfunction after BMT.

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

SummaryChronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon α. However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14+ and CD33+ cells but to a reduction in CD34+ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14+ and CD33+ as well as CD34+ cells but in a significant increase in CD3+ and CD56+ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation

SummaryBetween 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0° to II° occurred in 80% of our patients, and GvHD III° and IV° in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III° and IV° compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.