Ulrich R. Hengge

Incidence and prevalence of neurological disorders associated with HIV since the introduction of highly active antiretroviral therapy (HAART).

OBJECTIVE To determine the change of incidence and prevalence of neurological disorders caused by the human immunodeficiency virus (HIV) and opportunistic infections in HIV positive patients under treatment since the introduction of highly active antiretroviral therapy (HAART). METHODS The data of all HIV infected patients were retrospectively analysed, who were examined in the HIV outpatients clinic of the neurological department of the University Clinic Essen between 1995 and 1998 (n=563, total number of visits=735). Data from identified patients were divided into two groups according to the time of examination from 1995 to 1996 (334 visits) and from 1997 to 1998 (401 visits). The incidence and prevalence of neurological disorders were statistically compared between both time intervals. RESULTS Significantly more patients received HAART in 1997–8 (p<0.001) and mean CD4+ cell count was significantly higher in 1997–8 (p<0.001). The prevalence of HIV associated dementia and HIV associated polyneuropathy were significantly lower in 1997–8 (both: p=0.02) and the incidence of toxoplasma encephalitis decreased from 5.7% in 1995–6 to 2.2% in 1997–8 (p=0.015). Based on the small number of patients significant changes in HIV associated myopathy, progressive multifocal leukoencephalopathy, cryptoccocal meningitis, and cytomegalovirus-encephalitis could not be detected. CONCLUSION The prevalence of the most frequent HIV associated neurological disorders and incidence of toxoplasma encephalitis decreased since the introduction of HAART. This may be due to the improvement of immunostatus by HAART as demonstrated by the higher CD4+ cell count in the later time interval. Direct antiretroviral effects within the nervous system may be considered causative as well. The prevalence and incidence of HIV associated neurological disorders and opportunistic CNS infections decreased after introduction of HAART.

Expression of naked DNA in human, pig, and mouse skin.

The insertion and expression of genes in the epidermis may have a variety of therapeutic uses, including the treatment of skin diseases. Here we show that when both human skin organ cultures and human skin grafts on immunocompromised mice are injected with naked DNA, the DNA is taken-up and genes are expressed in the epidermis in a manner similar to both pig skin injected in vivo and injected pig skin organ cultures. In contrast, DNA injected into mouse skin is expressed not just in the epidermis, but also in the dermis and underlying fat and muscle tissue, and is expressed at lower levels. These findings suggest that genes can be expressed in human skin, after injection of naked DNA, and indicate that pig skin is an appropriate model for the study of DNA uptake and gene expression in human skin. The organ cultures of human and pig skin may be useful in understanding how naked DNA is internalized and expressed after in vivo injections. Additionally, skin obtained from patients with skin disease may be studied as skin grafts and organ cultures to help optimize genetic approaches for the treatment of skin diseases prior to clinical trials, by determining if the injected gene can provide a therapeutic benefit.

Scabies: a ubiquitous neglected skin disease

Scabies has been a scourge among human beings for thousands of years. Its worldwide occurrence with epidemics during war, famine, and overcrowding is responsible for an estimated 300 million people currently infested. Scabies refers to the various skin lesions produced by female mites, and their eggs and scybala that are deposited in the epidermis, leading to delayed-type hypersensitivity reaction. Recent immunological findings such as cross-reactivity with house dust mite allergens and an altered T-helper-1/T-helper-2 pattern contribute to a better understanding of the pathomechanism. Furthermore, progress in molecular biology and cloning of relevant antigens could enable the development of a diagnostic ELISA system and candidate vaccines in the near future. Typical and atypical clinical presentations with pruritus as a hallmark of scabies occur in young, pregnant, immunocompromised, and elderly patients and include bullous and crusted (Norwegian) manifestations as well as those masked by steroid use (scabies incognito). This article reviews scabies management strategies in developed countries and resource-poor communities as well as typical complications, including the emergence of resistance and drug-related adverse events. Other problems such as post-scabies eczema and reinfestation, and newer treatments such as ivermectin are also discussed.

Update on Kaposi's sarcoma and other HHV8 associated diseases. Part 2: pathogenesis, Castleman's disease, and pleural effusion lymphoma

The pathogenesis of Kaposis sarcoma (KS) is better understood since the identification of the novel human herpesvirus 8 (HHV8), which can be found in all forms of KS. Viral oncogenesis and cytokine-induced growth, as well as some states of immunocompromise, contribute to its development. Several virally encoded genes--eg, bcl-2, interleukin 6, cyclin D, G-protein-coupled receptor, and interferon regulatory factor--provide key functions on cellular proliferation and survival. Growth promotion of KS is further stimulated by various proinflammatory cytokines and growth factors such as tumour necrosis factor a, interleukin 6, basic fibroblast growth factor, and vascular endothelial growth factor, resulting in a hyperplastic polyclonal lesion with predominant spindle cells derived from lymphoid endothelia. HHV8 has recently been discovered to escape HLA-class-I-restricted antigen presentation to cytotoxic T lymphocytes by increasing endocytosis of MHC class I chains from the cell surface, thus enabling latent infection and immune escape in primary and chronic infection. Multicentric Castlemans disease is a rare lymphoproliferative disorder of the plasma cell type, which has been reported in both HIV-seropositive and HIV-seronegative patients, and which frequently contains HHV8 DNA. Pleural effusion lymphoma, or body-cavity-based lymphoma, belongs to the group of non-Hodgkin B-cell lymphomas characterised by pleural, pericardial, or peritoneal lymphomatous effusions in the absence of a solid tumour mass. Pleural effusion lymphoma has an intermediate immunophenotype lacking B or T lymphocyte antigens and also belongs to the diseases associated with HHV8.

SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition.

The suppressors of cytokine signaling (SOCS) are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Recently, methylation of SOCS-1 and SOCS-3 has been implicated in the tumorigenesis of liver and lung cancer. This study was performed to elucidate the role of SOCS-1 and SOCS-3 in squamous cell carcinoma of the head and neck (HNSCC) and its precursor lesions. HNSCC of 94 patients and corresponding normal mucosa, lymph node metastases as well as 16 high- and 21 low-grade squamous cell dysplasias were studied by using methylation-specific PCR (MSP) for the SOCS-1 and SOCS-3 promoter after microdissection. The presence of SOCS-3 mRNA transcripts was confirmed by semiquantitative real-time PCR, and the SOCS-3 protein was analysed immunohistochemically. SOCS-3 hypermethylation was found in 85/94 HNSCC (90%) and in 10/16 high-grade and 9/21 low-grade dysplasias (63 and 43%, respectively). SOCS-1 promoter hypermethylation was detected in 10/94 HNSCC samples (11%) and in 2/16 high-grade and 1/21 low-grade dysplasias (13 and 5%, respectively). Lymph node metastases exhibited an identical methylation status as the primary tumors. Methylation of the SOCS-3 promoter correlated with downregulation of SOCS-3 transcripts and protein expression in these tumors and various cell lines. In the cell lines tested, SOCS-3 and SOCS-1 transcripts increased upon treatment with the demethylation compound 5-aza-2-deoxycytidine (5-AZA-DC). Overexpression of wild-type SOCS-3 in carcinoma cells with methylated SOCS-3 resulted in the induction of apoptosis and growth suppression as well as downregulation of STAT3, bcl-2 as well as bcl-xL. Our data suggest that promoter methylation and subsequent transcript downregulation of SOCS-3 transcripts and, to a much lesser extent, SOCS-1 are involved in the multistep carcinogenesis of HNSCC. During its involvement in tumor growth, restoration of SOCS-3 may hold treatment potential for HNSCC.

Randomized, controlled phase II trial of subcutaneous interleukin-2 in combination with highly active antiretroviral therapy (HAART) in HIV patients

Objective:To determine the immunological, virological and clinical effects of subcutaneous IL-2 in 44 HIV-patients in conjunction with pre-existing tri-therapy (zidovudine, 3TC, saquinavir). Design:Partially randomized, controlled, prospective trial. Setting:Single center study at tertiary care center. Patients:Sixty four patients (CD4 count 200–500 × 106/l). Intervention:Fourty four patients were randomized to receive 5-day cycles of IL-2 (9 Mio IU/d) every 6 weeks (Group A) or whenever the CD4 cell count dropped below the 1.25-fold of baseline (Group B), whereas 20 control patients received the same HAART without IL-2. Outcome measures:The optimal individual treatment interval and the immunological and virological effects of subcutaneously administered IL-2 were analysed. Importantly, the level of cellular in vivo immunity and the frequency of dermatological marker diseases and infectious complications were assessed. Results:IL-2 was well tolerated although fever, influenza-like symptoms and indurated injection sites were commonly encountered. After 1 year of IL-2, there was a median increase of more than 100 × 106/l CD4 cells in both IL-2 groups in contrast to the controls (P < 0.01, 0.01 and not significant). The median HIV load did not increase either in plasma or in lymph nodes. Lymphocyte activation decreased as assessed by MHC class II (P < 0.001), CD25 (P < 0.001) and CD38 expression (P < 0.005). Although delayed type hypersensitivity against common recall antigens increased in both IL-2 groups, it did not reach statistical significance. However, it is of note, that in 7 of 11 (63.6%) patients delayed type hypersensitivity against recombinant HIV antigens improved significantly. Whereas there was no opportunistic infection in either IL-2 group, three cases of Kaposis sarcoma occurred in the controls. Dermatological indicator diseases (thrush, condyloma, herpes simplex) were found to occur more frequently in the control group. Conclusions:Subcutaneous IL-2 in addition to HAART was safe and led to sustained qualitative and quantitative immunological improvements in the majority of patients. Individualisation of therapy intervals further improved the efficacy and tolerance of IL-2.

Absence of Mutations of the BRAF Gene and Constitutive Activation of Extracellular-Regulated Kinase in Malignant Melanomas of the Uvea

The v-raf murine sarcoma viral homolog B1 (BRAF) gene, one of the human isoforms of RAF, is activated by Ras, leading to cooperative effects in cells responsive to growth factor signals. Recently, somatic missense mutations of the BRAF gene have been detected in more than 66% of malignant melanomas of the skin. We analyzed 42 malignant melanomas of the uvea, 3 corresponding liver metastases, and 10 cutaneous melanomas for possible BRAF mutations: after microdissection, mutation analysis of BRAF and KRAS was performed. The expression of extracellular-regulated kinase 1 and 2 (ERK1/2), an important downstream point of convergence in the Ras-RAF-MEK-Erk pathway, was analyzed immunohistochemically. Interestingly, we failed to detect activating BRAF mutations in uvea melanomas and their corresponding liver metastases. There were no mutations of BRAF in corresponding non-neoplastic uvea specimens, although we detected three BRAF mutations in sporadic cutaneous melanoma that led to a substitution of valine by glutamic acid at position 599 (V599E). KRAS mutations were detected in 1 of 10 cutaneous melanoma but not in uveal or metastatic melanoma. Despite the lack of activating mutations in the BRAF gene, we identified constitutively activated ERK in almost all (86%) uveal melanoma tissues tested but not in corresponding normal retina or uveal cells. Our data indicate that BRAF gene mutations are rare to absent events in uveal melanoma. The finding of activated Erk suggests a causative role for MAPK activation in uveal melanoma independent of activating BRAF or RAS mutations.

Oxymetholone promotes weight gain in patients with advanced human immunodeficiency virus (HIV-1) infection

The effect of the testosterone derivative oxymetholone alone or in combination with the H1-receptor antagonist ketotifen, which has recently been shown to block tumour necrosis factor alpha (TNF alpha), on weight gain and performance status in human immunodeficiency virus (HIV) patients with chronic cachexia was evaluated in a 30-week prospective pilot study. Thirty patients were randomly assigned to either oxymetholone monotherapy (n 14) or oxymetholone plus ketotifen (n 16). Patients receiving treatment were compared with a group of thirty untreated matched controls, who met the same inclusion criteria. Body weight and the Karnofsky index, which assesses the ability to perform activities of daily life, and several quality-of-life variables were measured to evaluate response to therapy. The average weight gain at peak was 8.2 (SD 6.2) kg (+ 14.5% of body weight at study entry) in the oxymetholone group (P < 0.001), and 6.1 (SD 4.6) kg (+10.9%) in the combination group (P < 0.005), compared with an average weight loss of 1.8 (SD 0.7) kg in the untreated controls. The mean time to peak weight was 19.6 weeks in the monotherapy group and 20.8 weeks in the combination group. The Karnofsky index improved equally in both groups from 56% before to 67% after 20 weeks of treatment (P < 0.05). The quality of life variables (activities of daily life, and appetite/nutrition) improved in 68% (P < 0.05) and 91% (P < 0.01) of the treated patients respectively. Oxymetholone was safe and promoted weight gain in cachectic patients with advanced HIV-1 infection. The addition of ketotifen did not further support weight gain. These results suggest the need for a randomized, double-blind, placebo-controlled multicentre trial.

Mutations of BRAF and KRAS2 in the development of Barrett's adenocarcinoma

Activation of the Raf/MEK/ERK (MAPK) signal transduction cascade by RAS mutations has been found in a variety of human cancers. Mutations of BRAF provide an alternative route for activation of this signalling pathway. To determine the role of mutations in BRAF and KRAS2 in the neoplastic progression of Barretts adenocarcinoma, we analysed both genes for common mutations. After microdissection, DNA of 19 Barretts adenocarcinomas, 56 Barretts intraepithelial neoplasias (n=29 low-grade intraepithelial neoplasia (LGIN) and n=27 high-grade intraepithelial neoplasia (HGIN)), 30 Barretts mucosa without neoplasia and normal squamous, as well as gastric epithelium, were analysed for BRAF and KRAS2 mutation. Activating BRAF mutations were identified in 2/19 Barretts adenocarcinomas (11%) and in 1/27 HGIN (4%). KRAS2 mutations were found in four out of 19 (21%) Barretts adenocarcinomas examined and in three cases of HGIN (11%). In LGIN as well as in normal gastric or oesophageal mucosa, neither BRAF nor KRAS2 mutations were detected. All lesions with KRAS2 mutations had an intact BRAF gene. The status of mismatch-repair proteins was neither related to BRAF nor KRAS2 mutations. These data indicate that RAS or BRAF mutations are detected in about 32% of all Barretts adenocarcinomas. We conclude that the disruption of the Raf/MEK/ERK (MAPK) kinase pathway is a frequent but also early event in the development of Barretts adenocarcinoma.

Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors

Previously, the RASSF1A, BLU and SEMAPHORIN 3B (SEMA3B) candidate tumor suppressor genes on chromosome 3p21.3 were found to be inactivated and downregulated by genetic and epigenetic changes in lung cancer. We analyzed the methylation status of RASSF1A, BLU and SEMA3B in 35 hepatocellular carcinomas (HCCs) and 15 cholangiocarcinomas (CCs) by methylation‐specific PCR and loss of heterozygosity (LOH) at 3p21.3 after microdissection. The presence of mRNA transcripts was confirmed by semiquantitative PCR. SEMA3B hypermethylation was found in 29/35 HCCs (83%) and in all (15/15) patients with CC. BLU promoter hypermethylation was detected in 7/35 (20%) HCCs and 3/15 (20%) CCs. In 2 corresponding specimens of hepatitis B virus‐related liver cirrhosis, BLU methylation was also observed, but not in uninvolved normal liver tissue. RASSF1A was methylated in 21/35 HCCs (60%) and in 10/15 CCs (67%). LOH at 3p21.3 occurred in 8/35 (23%) HCCs and 3/15 (20%) CCs. The presence of hypermethylation was statistically associated with LOH of SEMA3B and correlated with downregulation of mRNA transcripts. SEMA3B transcripts increased upon treatment of HCC cell lines with the demethylation compound 5‐aza‐2‐deoxycytidine. In conclusion, our data indicate that 2‐hit gene silencing of SEMA3B through epigenetic changes and allele loss is a common and important event in the carcinogenesis of malignant liver tumors.