[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

OBJECTIVE: To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. METHODS: According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. RESULT: The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. CONCLUSION: LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

[The HIV infection - the limits of prevention concepts. Consideration with respect to responsibilities incumbent on the infected individual, politics and society at large]. / HIV-Infektion - Grenzen der Präventionskonzepte. Uberlegungen zur Verantwortung der Betroffenen, der Politik und der Gesellschaft.

Despite the introduction of campaigns to prevent the continued spread of HIV/AIDS in Germany, the number of annual firsttime HIV-diagnoses is continuing steadily. The concepts behind the current campaigns are largely based on models of New Public Health, of which social learning strategies are an essential element. The established personal and individual rights should be unimpeachable but the right not to know the status of HIV infection should be questioned for those people who spread their HIV infection intentionally and wilfully. Confronted with more than 10,000 people in Germany unconscious of their HIV infection, easy access to HIV testing and access of opportune therapy should be offered with the goal of reducing the number of new infections. Expanded strategies on the responsibility to one's personal health and that of the partner, understandable and adapted to special groups of the society, should be established and maintained at a high level of awareness. All measures must be performed voluntarily.

Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis.

OBJECTIVES: To report the successful induction of remission with the monoclonal anti-CD20 antibody rituximab in a patient with hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis and a non-Hodgkin's lymphoma (NHL) resistant to previously advocated conventional treatments. CASE REPORT: The patient was a 45 year old woman with HCV associated cryoglobulinaemic vasculitis, with purpura, arthralgia, constitutional symptoms, and a polyneuropathy. A malignant NHL was found as underlying lymphoproliferative disease. At this stage the disease was refractory to interferon alpha2b and ribavirin and to subsequent immunosuppressive treatment with cyclophosphamide. Six rituximab infusions targeting the CD20 antigen on cells of the B cell lineage induced remission of the vasculitis. Bone marrow biopsy disclosed absence of the NHL. Remission has subsequently been maintained and HCV eliminated with the new pegylated interferon alpha2b and ribavirin for nearly one year. CONCLUSIONS: Transition of the underlying "benign" lymphoproliferative disease to a malignant lymphoma may result in difficult to treat HCV associated cryoglobulinaemic vasculitis. Rituximab offers a new possibility for inducing remission in refractory HCV associated cryoglobulinaemic vasculitis and the lymphoproliferative disorder. After remission, HCV may subsequently be eliminated with pegylated interferon alpha2b and ribavirin.

High homologous nucleotide to GBV-C was amplified from DNA of MT2 and HeLa cells and PBMC of human and chimpanzee.

AIM: To determine whether the nucleotide sequences homologous to GBV-C genome exist in the DNA from cell lines of human origin and from peripheral blood mononuclear cells (PBMC) of human and chimpanzee. METHODS: DNA of MT2 cell, HeLa cell, and PBMC from six human and one chimpanzee were prepared by using PCR Temperate Preparation Kit. All of the DNA preparations were digested with DNase-free RN ase and then were extracted by phenol-chloroform method. By using these DNA as templates, direct nested-PCR (dPCR) respectively amplified with GBV-C 5'-NCR and NS3 primers were carried out. Nucleotide sequences of the dPCR products were analyzed and positions in chromosomes of the amplification fragments were detected by using primed in situ (PRINS) sequence-specific labeling with fluorescein. RESULTS: DNA fragments amplified with GBV-C 5'-NCR primers were obtained from the DNA of MT2 and HeLa cells and the DNA in 4 of 6 human PBMC samples. DNA fragments amplified with GBV-C NS3 primers were obtained from MT2 and HeLa cells and the DNA in 5 of 6 human PBMC samples and one chimpanzee PBMC sample. The homology of the nucleotide sequences from these amplification products compared with the reported GBV-C genome sequence was from 73.80 % to 79.15 %. Fluorescence staining spots by using PRINS were shown in the PBMC and their chromosomes with positive dPCR results. CONCLUSION: The nucleotide sequences with high homology to GBV-C genome at the 5'-NCR and/or NS3 region exist in the DNA of MT2 and HeLa cells and the PBMC DNA of human and chimpanzee. These sequences locate in the chromosomes of PBMC with positive dPCR results.

DNA of nonhuman primates harbors hepatitis C-virus-specific sequences of its 5'-non-coding region (5'-NCR).

The DNA from PBMCs of both hepatitis C virus (HCV)-positive patients and healthy HCV-negative human individuals tested thus far contains essential parts--up to 272/341 nucleotides--of the HCV 5'-non-coding region (5'-NCR). These findings bring up the question of the possible evolutionary background of these sequences. Therefore, using the same methodology, we looked for the same sequences in animals closely related to man, i.e., in nonhuman primates (two chimpanzees, one orang-utan, one Debrazza monkey, two New World monkey species and a prosimian). The DNA from PBMCs of the studied animals belonging to nonhuman primates contains essential parts--up to 272/341 nucleotides--of the HCV 5'-non-coding region (5'-NCR). A common sequence of 82 nucleotides is contained in the DNA of all the tested animals but only the chimpanzee's DNA harbors the same, longer sequence region of 272/341 nucleotides of the 5'-NCR found in human DNA. The results may provide a clue as to the possible origin of parts of the IRES containing sequence area of the HCV.

Hepatitis C virus-specific DNA sequences in human DNA: differentiation by means of restriction enzyme analysis at the DNA level in healthy, anti-HCV-negative individuals.

This study aimed to look for further HCV-specific sequences at the DNA level of healthy, HCV-negative individuals. Here, the sequence section from nt 57 to nt 328 within the 5'-NCR was assayed. Different combinations of primers were used in the nested PCR without a preceding reverse transcriptase step, resulting in fragments of the expected molecular weight size and also shorter and longer ones. It shows that the major part of the 5'-non-coding region (5'-NCR) of the hepatitis C virus genome is present in the DNA fraction from peripheral blood mononuclear cells (PBMCs) of healthy, anti-HCV-negative individuals tested. Furthermore we designed experiments to prove the specificity of these findings, by using restriction enzymes for digest assays of the target DNA before PCR (pre-PCR digest) and of the products after PCR (post-PCR digest). In conclusion, our study indicates that the main part of the internal ribosome entry site (IRES) structure of HCV at least is contained in the DNA of the individuals tested.

The GOR47-1 sequence in human DNA encoding for a potential autoantigen in connection with hepatitis C--a sequence not only reserved for humans.

The sequence 'GOR47-1' is a consistent part of human DNA; the expressed polypeptide of it 'GOR' is accepted to be an autoantigen, and the anti-GOR an autoantibody. However, GOR47-1 was originally isolated through a cDNA clone from blood of a chimpanzee. This animal belonged to a series of chimpanzees, in which human plasma of a patient with non-A, non-B hepatitis had been passaged. To date, nothing is known how it is that this 'sequence GOR47-1' without recognizable self-replicating properties and allocated to the human genome could be isolated from a chimpanzee plasma. The aim of this study was to detect by polymerase chain reaction GOR47-1 sequences in healthy, anti-HCV-negative humans, HCV-positive patients, chimpanzee, snake, and in maize and tobacco plants. The GOR47-1 sequence is present not only in human DNA but also with a high degree of homology in chimpanzee DNA. Essential parts of this sequence are also present in DNA of a snake and the two plants listed above. Our findings reveal that the GOR47-1 sequence isolated from a chimpanzee was probably of the chimpanzee origin. This fact has not yet been considered up until now, when discussing the role of GOR/anti-GOR in humans particularly suffering from chronic hepatitis C.

Supplementary anti-hepatitis C virus (HCV) testing with 2nd and 3rd generation recombinant immunoblot assay and matrix applied to enzyme immunoassay positive sera and comparison with HCV-RNA detection.

A series of 118 serum samples tested positive in the anti-hepatitis C virus (HCV) 2nd-generation enzyme immunoassay (EIA-2), and indeterminate (93 samples) or negative (25 samples) in the supplementary 2nd-generation recombinant immunoblot assay (RIBA-2). These sera were further evaluated by three additional tests: 3rd-generation RIBA (RIBA-3), MATRIX, and AMPLICOR HCV-PCR. For the 93 RIBA-2 indeterminate serum samples, the results of the immunoassays had a concordance of 69%. Twenty-one and 34 samples remained anti-HCV indeterminate in the RIBA-3 test and the MATRIX, respectively. Among the 25 RIBA-2-negative samples, only seven samples remained anti-HCV negative, while five samples tested anti-HCV positive in both RIBA-3 and MATRIX. The reactivity of the RIBA-3 antigen NS5 was not crucial for the result of any sample. Positive to negative contradictions between the results of MATRIX and RIBA-3 were never observed. Altogether, the MATRIX tested a significantly lower number of samples anti-HCV negative than did the RIBA-3. HCV RNA was detectable in 54/93 RIBA-2 indeterminate and 7/25 RIBA-2 negative samples. High percentages of PCR positive results among RIBA-3-indeterminate and among MATRIX-indeterminate samples indicate an increased possibility of detecting HCV RNA if at least one antigen is reactive. The type of antigen, the pattern of antigen reactivity, or the level of reactivity had no prognostic value in predicting the presence of HCV RNA. Our findings show the necessity of being cautious in the interpretation of RIBA-2-negative results.

Early T-cell apoptosis and Fas expression during antiretroviral therapy in individuals infected with human immunodeficiency virus-1.

Several lines of evidence suggest that Fas-mediated apoptosis is involved in the CD4 T-cell depletion in human immunodeficiency virus-1 (HIV-1) infection. To investigate this, we studied changes in peripheral blood, early T-cell apoptosis and Fas expression after initiation of antiretroviral therapy (ART) in 18 HIV-1-infected individuals. Flow cytometric analysis was performed with Apostain and CD4, CD8 and Fas staining. Fas expression was quantified by standardized beads. The levels of CD4 and CD8 T cells with early apoptosis were increased comparably in HIV-1-infected individuals. Despite elevated CD4 T cell counts, no decline in early T-cell apoptosis could be detected during the first 8 weeks of ART. However, after 26 weeks of ART in five patients that showed a sustained reduction of viral replication there was a marked decrease in T cells with features of early apoptosis. No difference was found for Fas expression on early apoptotic T cells. Fas expression on CD4 and CD8 T cells was reduced after initiation of ART; this was independent of the CD4 T-cell trend and indicates that the immediate CD4 T-cell expansion during ART is probably not the result of a decreased rate of early apoptosis among peripheral blood CD4 T cells. However, preliminary data imply a long-term reduction of early T-cell apoptosis and Fas expression in patients who show a sustained reduction of viral replication.

[HBV-DNA positive findings in HBsAg negative blood donors and patients]. / HBV-DNA-positive Befunde bei HBsAg-negativen Blutspendern und Patienten.

There have been repeated discussions as to whether the implementation of anti-HBc screening of blood donations in Germany would be useful. We present several cases of HBsAg-negative patients and blood donors in whose plasma HBV-DNA was mainly found only after enrichment of virus particles by ultracentrifugation. In the case of a seroconverting blood donor, 3 of 4 HBsAg tests could not detect HBsAg. 90% of our HBsAg-negative, but HBV-DNA containing samples (n = 10) were positive for anti-HBc. The 'nested' PCR without previous ultracentrifugation was positive in only under 30% of the samples. Ultracentrifugation is an expensive method and will not be practicable for the testing of all blood donations. We conclude that the HBsAg tests which are available should be subjected to improvement. In addition, the implementation of anti-HBc screening could decrease the risk of posttransfusion HBV infection more effectively than PCR testing from nonenriched serum.

Hepatitis C virus (HCV) specific sequences are demonstrable in the DNA fraction of peripheral blood mononuclear cells from healthy, anti-HCV antibody-negative individuals and cell lines of human origin.

No convincing support has been provided so far for the existence of extrahepatic hepatitis C virus particles that should correspond to the sometimes extremely high concentration of 'HCV-RNA' in serum or plasma. If a naturally occurring HCV-specific DNA were to be found, a concept for at least some phenomena in terms of the pathophysiology of HCV should become conceivable. DNA was extracted from peripheral blood mononuclear cells of eleven healthy, anti-HCV-negative individuals, including five long term blood donors, and cells from different cell lines. DNA was subjected to nested polymerase chain reaction omitting a reverse transcriptase step with primers of the 5'NC as well as part of the core region of HCV. Direct polymerase chain reaction, i.e. without a reverse transcriptase step, revealed HCV-specific sequences in the DNA fraction of peripheral blood mononuclear cells of different origin: healthy anti-HCV negative individuals, furthermore in HeLa and MT2 cells. The fragments found were of expected length as well as of shorter and of longer than expected length with respect to the sequence of the HCV genome framed by the primers applied. The results derived from additional hybridization, restriction endonuclase analysis, and sequencing demonstrated HCV-specific sequences in the expected fragments with both a high degree of homology and deletions, respectively, substitutions, as compared to a prototype strain. However, the longer than expected fragments also contained sequences not specific for HCV.

Fas (CD95) expression on CD4+ T cells from HIV-infected patients increases with disease progression.

Active T cell suicide (apoptosis) is supposed to be involved in the CD4+ T cell depletion in the course of HIV infection. We investigated the expression of the apoptosis-related antigen Fas on CD4+ T cells from 25 HIV-positive individuals (CDC I-III) and 8 HIV-negative controls by two-colour flowcytometry. In addition, we evaluated: total CD4 count, HIV p24 antigen concentration in serum after immune complex dissociation, and clinical course of infection in HIV-positive individuals. We found a significant increase in mean Fas expression on CD4+ T cells from HIV-positive individuals compared to HIV-negative individuals (85.84 +/- 14.92% vs. 64.28 +/- 7.59%, P < 0.001). Within the HIV-positive group the increase in Fas expression was correlated with the decline in CD4 count (r = -0.76, P < 0.001), p24 antigen concentration in serum, after immune complex dissociation (r = 0.67, P < 0.001), and CDC stage (r = 0.73, P < 0.001). The upregulation of Fas antigen on CD4 cells is associated with CD4 depletion and other virological and clinical marker of disease progression in HIV infection.

HIV p24 antigen concentration in serum of 11 anti-HIV 1-positive patients before and after immune-complex dissociation: a study of a 5-year period.

BACKGROUND: Determination of the p24 antigen of the human immunodeficiency virus type 1 (HIV 1) is widely used to monitor viral activity though it is well known that substructures with p24 specificity present in serum can be complexed with specific antibodies, thereby preventing them from being detected by regular p24 antigen detection assays. OBJECTIVES: To compare the regular assay for p24 antigen with a procedure that dissociates immune-complexes before determination of this antigen. STUDY DESIGN: Eleven HIV 1-infected patients were followed for up to 5 years in order to obtain continuity in terms of the development of the p24 antigen in comparison with other surrogate markers. RESULTS: The results show that even low concentrations of anti-p24 antibodies are able to complex p24 antigens, rendering them undetectable in the routine assay. p24 antigens became detectable only after dissociation of these immune complexes by acid treatment procedure. In most patients viral activity became demonstrable only after application of the dissociation procedure.

Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV-1 on the surface of HIV-1-infected H9-lymphocytes.

Immunofluorescence staining techniques at present, when applied to follow the expression of HIV-1-specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV-1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV-1-infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell-like size specific for HIV-1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen-specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen-specific cell-mediated immunity with full antibody-dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.

Immunological diagnosis in viral infections of the central nervous system: course of antibody titres against homo- and heterologous viruses.

In clinical cases suspected for viral encephalitis or meningoencephalitis, the estimation of virus-specific antibodies especially in liquor requires high sensitivity as well as specificity. With enzyme immunoassays the sensitivity in detecting antibodies has increased compared to e.g., complement fixation tests. This report concerns the determination of virus-specific antibodies with a commercial enzyme-linked immunosorbent assay (ELISA) in paired liquor/serum samples of four patients with encephalitis or meningoencephalitis. Up to six virus-specific antibodies of the IgG and IgM classes have been determined [herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus, mumps virus, measles virus, and rubella virus]. Additionally, serum samples from several patients suffering, or recovered from, diseases caused by HSV and VZV without CNS involvement have been included as controls. The results showed that besides the virus-specific antibody development (IgG and IgM) against the leading virus, i.e., principally concerned in the disease manifestation assumed to be primarily causing the disease, virus-specific antibodies of the IgG and IgM class against a heterologous virus (e.g., VZV) could also be measured with substantial titers. "Cross-reacting" antibodies to both HSV and VZV with the ELISA only appeared and were present in cases where the infection mainly affected the CNS: no such immunological "cross-reactivity" was observed in serum of individuals in "clinically silent" stages of both HSV and VZV infections. The same situation with no measurable "cross-reacting" antibodies was found in cases of acute HSV or VZV diseases where the CNS was not involved. These findings have been discussed with respect to the findings of common antigens, especially between HSV and VZV, and with respect to an unspecific stimulation of immunocompetent cells.

[Acute retinal necrosis]. / Akute retinale Nekrose.

The authors report on three patients with acute retinal necrosis who were treated with the virostatic agent Acyclovir and who underwent vitreoretinal surgery with silicone oil filling for total retinal detachment. In two eyes the retina was reattached, but useful vision was only preserved in one patient. Titers from blood and the vitreous, as well as microscopic findings in retinal biopsies, support the view that the necrosis is caused by a herpes simplex virus infection. After therapy with Acyclovir was instituted no further progression on the necrosis was observed. However, the development of retinal detachment could not be prevented. Early diagnosis and antiviral therapy are essential to improve the otherwise poor prognosis in this rare syndrome.

[Diagnostic value of lymphocyte differentiation in the early phase following kidney transplantation]. / Diagnostischer Wert der Lymphozytendifferenzierung in der Frühphase nach Nierentransplantation.

In 50 patients who had a renal transplantation, treated with ciclosporin, regular monitoring of lymphocyte subpopulations was undertaken prospectively to assess its value with respect to cellular rejection, herpes virus infection, and ciclosporin overdosage. Herpes virus infection was characterized by inversion of the T4/T8 ratio below 1.0 (sensitivity 90%, specificity 88%), caused by proliferation of the T8 subpopulation, which--compared with the findings in patients with rejection crises--was significantly raised (P less than 0.001). But such rejection crises could not be predicted from the T4/T8 ratio. Ciclosporin had no effect on the ratio, total lymphocyte count in this group being higher (P less than 0.002) than in patients with rejection.

[Specific antibodies to herpes simplex virus, cytomegalovirus and rubella virus: distribution in amniotic fluid and blood]. / Spezifische Antikörper gegen Herpes-simplex-Virus, Zytomegalie-Virus und Röteln-Virus: Verteilung in Fruchtwasser und Blut.

Fifty-nine unselected pregnant women were included in the study at the time of delivery. Immediately post partum maternal blood was obtained by venous puncture, and fetal blood from the umbilical cord. Amniotic fluid was obtained by amniocentesis during labor prior to rupture of the amniotic sac. Virus-specific rubella, herpes simplex and cytomegalo virus antibodies were determined with a commercial enzyme immune test. The virus-specific antibody titers are higher by a factor of 1.3 to 1.5 in the fetal than in the maternal serum. In contrast, the concentration of the corresponding antibodies in the amniotic fluid is approximately 5 titer levels (log 2) lower than in the maternal serum - regardless of the maternal antibody titer level. In view of its low concentration of antibodies the amniotic fluid can hardly be regarded as an "immunological barrier" if it serves as a "transport or distribution medium" in some virus infections. However, passive intra-amniotic immunization could be useful for combating certain virus infections during pregnancy, especially just before term.

[Modification of the incidence and course of CMV infections following kidney transplantation by passive immunization--initial experiences with a hyperimmunoglobulin]. / Beeinflussung von Inzidenz und Verlauf von CMV-Infektionen nach Nierentransplantation durch passive Immunisierung--erste Erfahrungen mit einem Hyperimmunglobulin.

In 45 recipients of a renal transplant the CMV-antibody titer was measured preoperatively (ELISA method). If possible, the donors were examined likewise. All seronegative recipients of grafts of a seropositive donor were immunized passively with a CMV-hyperimmunoglobulin for 6 months (2 ml/kg bw in 3 weeks intervals). 7 patients showed that constellation, and they were treated. 4 of them demonstrated neither serological nor clinical signs of a CMV-infection at any time. In 3 patients an infection was found serologically, but only 2 showed concomitant clinical symptoms. No serious complications (pneumonia etc.) were seen. One year later all patients are doing well with a functioning graft. For this reason we think the passive immunization of a seronegative recipient of a graft from a seropositive or unexamined donor to be advisable.