Comparative study of delayed hypersensitivity skin reactions and antibodies to human papilloma (HPV)

Purified human papilloma virus (HPV) was prepared from plantar warts, inactivated by formalin, adjusted to 10¹⁰ particles per 0·1 ml and used in intradermal tests (IDT) on 120 patients having warts at that time, or having had warts in the past, together with sixty-three controls. Antibodies were evaluated in the sera from most of the patients by the indirect immunofluorescence (IF) test before and after the skin tests. The results showed a specific delayed-hypersensitivity reaction (DHR) to HPV, especially in patients with `regressing' or `past' warts (76%). DHR was most frequent in patients with a wart of 6 months–2 years duration (77·0–68·8%) and persisted much longer than antibodies, which disappeared with the passage of time. The incidence of antibodies was much higher after IDT in patients with a positive reaction, thus suggesting a booster effect.     

Langerhans cells and epithelial cell modifications in cervical intraepithelial neoplasia: correlation with human papillomavirus infection

The study of a series of 18 cervical intraepithelial neoplasia (CIN) grade II and III was aimed at determining the distribution and phenotype of immunocompetent cells (Langerhans cells, T and NK cells) and the alteration in the expression of EGF receptors and beta 2-microglobulin in correlation with human papillomavirus (HPV) infection (viral antigen and DNA typing with biotinylated probes). These lesions were characterized by a reduced number of Langerhans cells and a dense infiltrate. HPV infection did not induce HLA-DR expression in the infected epithelial cells. We observed an enhanced expression of epidermal growth factor (EGF) receptors by epithelial cells and a reduced beta 2-microglobulin reactivity by both epithelial and immunocompetent cells. Most of CIN showed foci of infected cells. No significant differences were observed in immunological markers of CIN harboring benign HPV 6/11 DNA or oncogenic HPV 16/18 DNA. Viral antigen was not detected in these lesions. These changes in the epithelial cells of CIN and their microenvironment associated to the lack of HLA-DR expression in the infected cells hamper the squamous epithelial cells to function as antigen presenting cells. This may facilitate a decrease in the immunological surveillance and may contribute to the severity of such lesions.     

Virus expression EGF and transferrin receptors in human papillomas

Summary Thirty five non regressing cutaneous and mucosal human papillomas were studied for the expression of EGF and transferrin receptors by indirect immunofluorescence on frozen sections. The lesions were also examined for the presence of human papillomavirus (HPV) DNA by in situ-hybridization with biotinylated probes and viral capsid antigen. The mapping of EGF and transferrin receptors was modified in cutaneous lesions with drastic viral cytopathic effects and was enhanced in mucosal lesions mainly in laryngeal papillomas, which are poor virus producers. The greatest increase in EGF and transferrin receptor reactivity was observed in the group of mucosal lesions in which viral DNA was more frequently detected than viral antigen. This suggests that viral DNA may play a role in basal cell stimulation. Moreover some of these lesions with dense inflammatory reactions showed DR antigen expression by epithelial cells. Our findings indicate that epithelial cell activation in papillomas might be modulated by other factors than HPV such as mediators of the local immune response.This work was supported by grants from Féderátion Nationale des Centres de Lutte Centre le Cancer, 1985 and Fondation Mérieux, Lyon, France     

Early events in the interaction of adenoviruses with HeLa cells. IV. Association with microtubules and the nuclear pore complex during vectorial movement of the inoculum

Only minutes elapse before adenovirus 5 (AV5) particles are transferred from the cell surface to the perinuclear site of uncoating. Morphological evidence suggesting that virions are moved vectorially along pathways provided by the microtubules is supported by experiments with vinblastine, which induces the formation of microtubular paracrystals (PC): intimate attachment of AV5 to PC was observed within intact cells or following isolation of virion-PC complexes and was inferred after mixing in vitro PC and AV5 suspensions. Together with data showing a delay in the initiation of the infectious cycle by vinblastine, these observations imply that when microtubules become concentrated within PC, normal transfer of the inoculum to the nucleus is, at least in part, affected. On the other hand, failure to interrupt appreciably the flow of AV5 to the nucleus by colchicine appears to be inconsistent with the above finding and may imply that intact microtubules are not involved in the vectorial movement. The nuclear pore complex (NP) was further identified as the specific ultimate site of intracellular AV binding since (a) colchicine elicited the formation of NP-like organelles in the cytoplasm to which inoculum virions are bound; (b) virions were isolated as part of a stable complex with NP on nuclei released from recently infected cells and, furthermore, the AV5-NP association survived isolation and purification of the nuclear envelopes. Application of p-hydroxymercuric benzoate at nontoxic concentrations suppressed both the uncoating and an ATPase activity of nuclear envelopes present at or near the NP. This result provides further circumstantial evidence in support of our previous hypothesis stating that AV uncoating at the NP is mediated by an ATPase.     

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.     

Long-term functional results of selective peripheral neurotomy for the treatment of spastic upper limb: prospective study in 31 patients

OBJECT: To manage refractory upper-limb spasticity, selective peripheral neurotomy (SPN) is proposed when the spastic muscles to be treated are under the control of a single or a few peripheral nerves. The aim of this study was to assess prospectively the long-term effects of SPN. METHODS: Thirty-one patients with disabling upper-limb spasticity were selected by a multidisciplinary team using clinical, analytical, and functional scales as well as nerve block tests for assessment. Sixty-four SPNs were performed at the level of the musculocutaneous (15 SPNs), the median (25 SPNs), and the ulnar (24 SPNs) nerves. Results of a long-term follow up (mean 4.5 years) showed statistically significant improvement on 1) analytical assessment (p < 0.01): resting position, active amplitude, and motor strength; 2) Ashworth Scale scoring (p < 0.01); 3) hand function assessment (p < 0.01); and 4) rating of daily activities. Four patients with severe painful spasticity experienced complete pain relief after surgery. On the basis of a Visual Analog Scale ranging from 0 to 100, the mean degree of patient satisfaction was 61.5. Complications occurred in five patients (15%): two postoperative hematomas, one (temporary) hypesthesia, and one transient paresia of the wrist and finger flexors. CONCLUSIONS: Selective peripheral neurotomy leads to long-term satisfactory improvement in function and/or comfort with a low morbidity rate in appropriately selected patients suffering from severe harmful spasticity of the upper limb that has been refractory to conservative therapy. Patients must be selected after complete assessment by a multidisciplinary team.     

Detection of multiple types of human papillomavirus in a giant condyloma from a grafted patient. Analysis by immunohistochemistry, in situ hybridisation, Southern blot and polymerase chain reaction.

Immunosuppressed patients such as transplant recipients are known to develop multiple lesions suggestive of human papillomavirus (HPV) infection. A giant anal condyloma was obtained from a transplant patient; several fragments taken from different areas were examined for the presence of HPV DNA using in situ hybridisation, polymerase chain reaction (PCR) and Southern blot. Typical koilocytes were seen in routinely stained tissue sections, suggesting an HPV infection; furthermore, group specific HPV antigen was detected in one of four frozen fragments. Different results were obtained by in situ hybridisation according to the fragment tested. HPV types 6/11 were detected in each of the five fragments, frozen or fixed in Bouin's or formalin solutions. However, the number of HPV DNA positive cells and the intensity of the reaction greatly varied with the specimen. HPV 16 and 18 probes also reacted positively with the sample fixed in formalin; a stronger signal was observed with HPV 18 in one large focus than with HPV 16. HPV type 5 was detected in a few isolated cells of two frozen fragments. With the Southern blot technique, the profile of an HPV 6/11 was seen only in one of two frozen fragments; in this case, the bands were intense. A slight positive reaction was also obtained in one frozen fragment with HPV 16 probe. Four frozen fragments were analyzed with PCR: HPV 6/11 was detected in each fragment; HPV 18 was detected in the four samples but with different intensities; HPV types 5 and 16 did not show any positive signal. In conclusion, the lesion is an example of infection with several HPV types, demonstrated by three different techniques. This suggests the need for careful dermatological or colposcopic follow-up of transplant recipients, in order to prevent possible malignant transformation of anogenital lesions.     

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.