Istvan Arany

A Randomized, Controlled, Molecular Study of Condylomata Acuminata Clearance during Treatment with Imiquimod

Imiquimod, an immune response modifier, has been demonstrated to be safe and effective in the treatment of external genital and perianal warts caused by human papillomavirus (HPV). To identify the molecular mechanism(s) by which condylomata acuminata clear during topical treatment with imiquimod, wart skin biopsies were taken from patients before treatment, at treatment week 6, and at the end of treatment. Tissues were analyzed for HPV DNA and for mRNA of several cytokines and HPV gene products. Wart clearance was associated with evidence of tissue production of interferon-alpha, -beta, and -gamma and tumor necrosis factor-alpha. Regression of warts was strongly associated with a decrease in HPV DNA and in mRNA expression for both early and late viral proteins. Thus, topical imiquimod treatment of anogenital warts led to significant increases in local production of multiple interferon mRNAs and a significant reduction in virus load as measured by decreases in HPV DNA and mRNA for early HPV proteins.

p66shc Inhibits Pro-survival Epidermal Growth Factor Receptor/ERK Signaling during Severe Oxidative Stress in Mouse Renal Proximal Tubule Cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66shc isoform can inhibit this p46/52shc function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser36 phosphorylation of p66shc. Isoform-specific knockdown of p66shc or mutation of Ser36 to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66shc to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser36 phosphorylation of p66shc and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser36 phosphorylation of p66shc or knocking down p66shc expression in vivo.

Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.

ObjectiveThis study determined whether genomic amplification of HER-2/neu or mutations of the p53 and ras genes were present in gastrinomas. Summary Background DataAmplification of HER-2/neu, a proto-oncogene related to the epidermal growth factor receptor, and mutation of the ras proto-oncogene and p53 tumor suppressor gene appear to play a role in the pathogenesis of some human cancers. Little is known about possible molecular alterations in gastrinomas, tumors that may be particularly virulent because of gastrin overproduction, resulting in the severe ulcer diathesis, the Zollinger-Ellison syndrome. MethodsThe differential polymerase chain reaction (PCR) procedure was used to detect amplification of the HER-2/neu gene in DNA samples from the novel human gastrinoma cell line (PT) and from paraffin-embedded samples of gastrinomas. Sequencing techniques were used to determine whether mutations of the p53 or ras (Ha-ras, N-ras, Ki-ras) genes were present. ResultsAmplification (> twofold) occurred in all gastrinoma tumor samples. Compared with normal pancreas or ileum, a 4− to 12-fold amplification of HER-2/neu was found in 3 gastrinomas, 3 to 3.3-fold in four samples and 2.1− to 2.4-fold in the remaining five tumors. A heterozygous point mutation in the p53 gene (codon 273) was found in a single sample; none of the gastrinomas contained a mutation of the ras genes. ConclusionsAmplification of the HER-2/neu gene, but not alterations of either p53 or ras, may be involved in the pathogenesis of gastrinomas. The unique PT cell line will be a useful model to further elucidate the molecular mechanisms that contribute to gastrinoma formation and growth.

Response of keratinocytes from normal and psoriatic epidermis to interferon-γ differs in the expression of zinc-α2-glycoprotein and cathepsin D

Psoriasis is a T cell‐mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc‐α2‐glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon‐γ (IFN‐γ), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN‐γ binding and signaling were attenuated in psoriasis: The IFN‐γ receptor, the signal transducer and activator of transcription STAT‐1, and the interferon regulatory factor IRF‐1 were strongly up‐regulated by IFN‐γ in normal keratinocytes, but not in psoriatic ones. IFN‐γ strongly up‐regulated the expression of the catalytic enzymes cathepsin D and zinc‐α2‐glyco‐protein in normal keratinocytes but down‐regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl‐2. We believe that the aberrant response to IFN‐γ plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.—Chen, S.‐H., Arany, I., Apisarnthanarax, N., Rajaraman, S., Tyring, S. K., Horikoshi, T., Brysk, H., Brysk, M. M. Response of keratinocytes from normal and psoriatic epidermis to interferon‐γ differs in the expression of zinc‐α2‐glycoprotein and cathepsin D. FASEB J. 14, 565–571 (2000)

Isoforms of cathepsin D and human epidermal differentiation

Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.

Status of local cellular immunity in interferon-responsive and -nonresponsive human papillomavirus-associated lesions.

Background and Objectives: Anogenital warts are caused by human papillomaviruses (HPVs), which should induce cellular immune responses in immunocompetent patients. However, the natural history of these warts shows considerable variation between persons, ranging from spontaneous regression to prolonged persistence. In addition, the efficiency of immunologically based modalities for the therapy of anogenital warts, such as interferon (IFN) treatment, is highly variable. Methods: Considering that preexisting conditions of the host are important factors in an appropriate immune response, the authors determined the pretreatment status of local cell‐mediated immune response to HPV infection by reverse transcription‐polymerase chain reaction in patients with condyloma acuminatum, who later received IFN treatment and responded well or poorly to that therapy. Results and Conclusions: The authors found that biopsies from nonresponders were depleted markedly in Langerhans cells, leading to decreases in major histocompatibility complex class II expression and, therefore, to diminished attraction of CD4+ T cells. An inappropriate major histocompatibility complex class I expression also was observed in those nonresponders with decreased CD8+ levels. The mRNA levels of cytokines (interleukin‐1a, interleukin‐1b, granulocyte‐macrophage‐colony stimulating factor, tumor necrosis factor that participate in immune responses were low in nonresponders. In contrast, responders demonstrated high macrophage‐natural killer cell (CD16‐positive) and activated CD4 (IL‐2, interferon gamma‐positive,TH1 cells) T‐cell recruitment against HPV‐infected keratinocytes, which is consistent with a delayed‐type hypersensitivity‐like cellular immune response. Lack of immune response in nonresponders appeared to correlate with high expression levels of the HPV E7 gene. These differences in local cellular immunity might determine the response rate of HPV‐infected cells to immunomodulatory therapies.

Human papillomavirus infections: epidemiology, pathogenesis, and therapy.

A bstractBackground: Human papillomaviruses (HPV) are common human pathogens and are classified into more than 80 different types. These viruses produce benign warts in many cases and aggressive squamous cell carcinomas in other cases. Objective: The goal of this review is to update the reader on the epidemiology, pathogenesis, and therapy of HPV infections. Nonanogenital warts are transmitted by skin-to-skin contact while anogenital warts are usually transmitted sexually. Both types of warts produce much morbidity but rarely undergo malignant transformation. They are commonly treated with surgical or cytodestructive therapy, but immunomodulatory agents, such as imiquimod, have been proven to be very effective in anogenital warts and are being evaluated in nonanogenital warts. Other types of HPV have marked oncogenic potential such that over 99% of all cervical cancers and over 50% of other anogenital cancers are due to infection with oncogenic HPV. Many cofactors, such as cigarette smoking, genetics, and helper viruses, have potential roles in HPV oncogenesis, but their relative contributions are poorly understood. Other control measures for warts and HPV-associated cancers are under study, but the greatest future potential may be from the development of prophylactic and therapeutic vaccines. Conclusions: Infection with HPV is very prevalent as are the clinical manifestations of this family of pathogens. Improved therapies for warts (e.g., imiquimod) have recently become available. Vaccines for HPV offer hope for future interventions for warts as well as for prevention of anogenital malignancies.

Chronic nicotine exposure exacerbates acute renal ischemic injury

Recent epidemiological reports showed that smoking has a negative impact on renal function and elevates the renal risk not only in the renal patient but perhaps also in the healthy population. Studies suggested that nicotine, a major tobacco alkaloid, links smoking to renal dysfunction. While several studies showed that smoking/chronic nicotine exposure exacerbates the progression of chronic renal diseases, its impact on acute kidney injury is virtually unknown. Here, we studied the effects of chronic nicotine exposure on acute renal ischemic injury. We found that chronic nicotine exposure increased the extent of renal injury induced by warm ischemia-reperfusion as evidenced by morphological changes, increase in plasma creatinine level, and kidney injury molecule-1 expression. We also found that chronic nicotine exposure elevated markers of oxidative stress such as nitrotyrosine as well as malondialdehyde. Interestingly, chronic nicotine exposure alone increased oxidative stress and injury in the kidney without morphological alterations. Chronic nicotine treatment not only increased reactive oxygen species (ROS) production and injury but also exacerbated oxidative stress-induced ROS generation through NADPH oxidase and mitochondria in cultured renal proximal tubule cells. The resultant oxidative stress provoked injury through JNK-mediated activation of the activator protein (AP)-1 transcription factor in vitro. This mechanism might exist in vivo as phosphorylation of JNK and its downstream target c-jun, a component of the AP-1 transcription factor, is elevated in the ischemic kidneys exposed to chronic nicotine. Our results imply that smoking may sensitize the kidney to ischemic insults and perhaps facilitates progression of acute kidney injury to chronic kidney injury.

Interferon treatment enhances the expression of underphosphorylated (biologically-active) retinoblastoma protein in human papilloma virus-infected cells through the inhibitory TGFβ1/IFNβ cytokine pathway

Interferons (IFN) regulate transcription of certain genes playing a role in cell proliferation. Targets of IFN action may include tumor suppressor genes such as the retinoblastoma (RB) gene and cytokines such as transforming growth factor beta 1 (TGF beta 1) and IFN beta which are inhibitors of epithelial cell proliferation. Using reverse transcription followed by PCR amplification, an increase of those growth inhibitory gene mRNA levels (TGF beta 1, IFN beta and RB) were found after interferon treatment in condylomas harboring non-oncogenic human papilloma virus (HPV 6/11) types, in an oncogenic HPV 16-containing cell line, and in a HPV negative, epidermoid carcinoma cell line. In addition, immunodetection by Western blot demonstrated a higher proportion of underphosphorylated (active form) retinoblastoma gene protein (pRB) after IFN treatment due to the decrease in the phosphorylating cdc2 kinase levels. Changes in the phosphorylation pattern of pRB together with the increased expression of those inhibitory genes represent a growth inhibited state in those cells as demonstrated by diminished c-myc expression. Since the extent of c-myc inhibition was significantly lower in the case of oncogenic HPV infection, a role of viral oncoproteins in abrogation of the antiproliferative effect of IFN therapy could be considered. These results demonstrate a new mechanism via which IFNs exert their antiproliferative effect on HPV-infected cells by affecting the expression and phosphorylation of the RB tumor suppressor gene, through the inhibitory TGF beta 1/IFN beta cytokine pathway.

Development of Human Papillomavirus Type 16 Associated Squamous Cell Carcinoma of the Scrotum in a Patient with Darier's Disease Treated with Systemic Isotretinoin

In contrast to squamous cell carcinoma of the penis, scrotal carcinoma has historically been associated with exposure to environmental or industrial carcinogens and has only rarely been correlated with human papillomavirus. We report on a patient with squamous cell carcinoma of the scrotum in which human papillomavirus type 16 was integrated into the tumor cell genome, suggesting a causal role of human papillomavirus in the development of squamous cell carcinoma of the scrotum. Other unique features of our case include the presence of Dariers disease, an uncommon genodermatosis, and treatment with oral retinoids, which have prophylactic value in the prevention of cutaneous malignancies.