The COLIPA in vitro UVA method: a standard and reproducible measure of sunscreen UVA protection.

There is a continuing need to measure and communicate reliably the UVA protection offered by commercial sunscreens. To that end, the COLIPA (European Cosmetics Trade Association) 'In Vitro Sun Protection Methods' group has developed a new in vitro method for measuring UVA protection in a standardized, reproducible manner. The method is based on in vitro UV substrate spectrophotometry and convolution of resulting absorbance data with the action spectrum for the in vivo Persistent Pigment Darkening (PPD) endpoint to provide an in vitro UVA protection factor (UVAPF) which is correlated with an in vivo measure. This method has been published as a COLIPA guideline, used currently in European geographies for testing and labelling sunscreen products. This article summarizes two 'ring' studies, involving eight separate testing laboratories, which both defined critical parameters for the method and validated it. In Ring Study 1, eight laboratories tested the in vitro UV transmission of a total of 24 sunscreens and, from the data, a unit dose of UVA (D(0) of 1.2 J cm(-2)) was defined to provide a single irradiation step which, by taking into account potential sunscreen photo-instability, gave the closest agreement with in vivo UVAPF values. In Ring Study 2, eight laboratories tested the in vitro UV transmission of a total of 13 sunscreens using this single irradiation step and established a very good correlation (r(2) = 0.83; slope = 0.84, P < 0.0001) between resulting in vitro UVAPF values and corresponding values derived from the in vivo PPD method. This new method, therefore, can be used to provide a reliable in vitro metric to describe and label UVA efficacy in sunscreen products, in line with the EU Commission recommendation 2006/247/EC.

Two ceramide subfractions detectable in Cer(AS) position by HPTLC in skin surface lipids of non-lesional skin of atopic eczema.

The non-involved skin of atopic eczema (NEAE) is characterized by severe dryness and an impaired barrier function of the stratum corneum as indicated by an increased transepidermal water loss. Previous studies have demonstrated that this barrier impairment coincides with marked alterations in the amount and composition of stratum corneum ceramides. The aim of this study was to identify specific alterations in NEAE that may be used in the diagnosis of the atopic eczema. Using a classical procedure for high performance thin layer chromatography we could confirm earlier results: apart from Cer(EOH), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to 6-hydroxy-4-sphingenine (H), the quantities of all ceramide fractions were significantly decreased. Furthermore, Cer(EOH)/Certotal was significantly increased, whereas the percentage of Cer(EOS), which contains sphingosine (S), and Cer(NP), which contains non-hydroxy fatty acid (N) amide-linked to phytosphingosine (P), were significantly decreased. Using a modified procedure for high performance thin layer chromatography we could demonstrate the formation of a double peak in the position of Cer(AS), which contains alpha-hydroxy fatty acid (A), in lipids of NEAE. The subfractions of the double peak comprised 15% and 12% of Certotal. MALDITOF mass spectrometry suggested that the double peak was formed by a homologous series of mono-hydroxylated and mono-unsaturated ceramides of different chain length, e.g., Cer(AS) subfractions containing either (C16,18) or (C22,24,26) alpha-hydroxy fatty acids. In contrast, in normal skin a single peak in Cer(AS) position, which comprised 22% of Certotal, was mainly formed by the long chain subfraction. In some cases this single peak displayed a small shoulder at its right flank, but never showed a clear peak separation when developed with NEAE samples. Furthermore, even in senile xerosis, or in either non-involved skin of psoriasis or seborrhoic eczema, only a single peak occurred in Cer(AS) position. Accordingly, the double peak might be specific for NEAE and turn out to be a marker for atopic eczema.

Anal cloacogenic and squamous carcinomas. Comparative histologic analysis using in situ hybridization for human papillomavirus DNA.

In order to evaluate the morphologic and possible etiologic distinctions between anal cloacogenic and squamous carcinomas, we performed histologic examination and in situ hybridization for human papillomavirus (HPV) DNA on anal canal and anal verge carcinomas from 37 patients. Twenty-one neoplasms were invasive or in situ squamous carcinomas, 14 were invasive cloacogenic carcinomas, and two were unclassified. In situ hybridization was positive for HPV types 16/18 in 12 cases and for types 6/11 in two cases of anal squamous carcinoma (67% HPV positivity overall). All 14 cases classified as anal cloacogenic carcinoma were negative for HPV DNA by this technique. One of the two unclassified carcinomas was positive for type 16/18 DNA. We conclude that anal cloacogenic and squamous carcinomas are histologically similar but distinct neoplasms. Differential expression of HPV DNA in these lesions may be a manifestation of separate mechanisms of pathogenesis, or it may be due to varying degrees of tumor cell differentiation.

Chronic human skin graft rejection in severe combined immunodeficient mice engrafted with human PBL from an HLA-presensitized donor.

Mice with severe combined immunodeficiency (C.B-17 scid [SCID]) accepted xenografts of adult human peripheral blood leukocytes injected intraperitoneally as evidenced by production of human immunoglobulin (IgG and IgM), and circulation of human leukocytes in peripheral blood. SCID mice also accepted human split-thickness skin xenografts. Passenger leukocytes present in small numbers in such skin grafts could also recirculate in host peripheral blood and make detectable levels of human immunoglobulin. To test the immunocompetence of the transferred human PBL, SCID mice received a human skin xenograft from a second donor (HLA-mismatched with the PBL donor) either before (n = 6) or after (n = 23) xenografting of PBL. Skin was monitored daily for signs of rejection, and rejection was scored by histology 3-4 weeks after the second graft (PBL or skin) was placed. Of 19 SCID injected with PBL from an HLA presensitized patient (L.G.), 7/19 (37%) rejected a subsequent HLA-mismatched skin xenograft. Two of six SCID (33%) rejected a previously established skin xenograft when PBL were administered afterward. The rejection of the human skin was chronic, of relatively late onset (3-4 weeks), and was characterized grossly by contraction, glassy surface, and thickening. Histopathologic examination showed lymphocyte infiltration into the dermis with endothelial cell cuffing and destruction of capillaries, as well as lymphocyte tagging of the basal epidermis, hyperkeratosis, lymphocyte exocytosis and single epidermal cell necrosis. Immunostaining with monoclonal antibody to human CD2 or mouse CD3 revealed that human, but not mouse T lymphocytes were tagging the dermis/epidermis junction and infiltrating the epidermis of rejecting skin grafts. We conclude that a form of human skin graft rejection may be reproduced in an SCID mouse. The immune status of the transferred cells (sensitized vs. normal) and the lymphocytes ability to recirculate in SCID peripheral blood appear to be factors limiting the rejection process.

Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.

Colonic lymphocytosis in patients with celiac sprue.

We examined colonic biopsies from 39 patients with clinical and small bowel biopsy changes of celiac sprue. In 12 of 39 patients (31%), striking lymphocytic infiltration of the superficial colonic epithelium and chronic inflammation of the lamina propria were identified. These 12 cases had a mean of 30.4 lymphocytes per 100 superficial colonic epithelial cells, compared with means of 8.4 in sprue cases without colonic epithelial lymphocytosis, 4.8 in normal controls, and 32.4 in nine cases of lymphocytic colitis without concurrent celiac sprue. No case showed subepithelial collagen layer thickening. Four patients with celiac sprue and colonic lymphocytosis also had gastric biopsies; two showed gastric lymphocytosis. Intraepithelial lymphocytes at all sites were positive for the T-cell marker MT-1. These findings indicate that sprue-associated colonic lymphocytosis and lymphocytic colitis are histologically, quantitatively, and immunohistochemically indistinguishable, that the epithelial T-cell infiltration of celiac sprue occurs in glandular mucosa at all levels of the gastrointestinal tract, and that colonic subepithelial collagen deposition in patients with celiac sprue is an infrequent occurrence. These findings also suggest that gastrointestinal epithelial T-cell infiltration may be an immunologic response that is common in individuals sensitized to absorbed lumenal antigens, and that colonic lymphocytosis may occur as a response to a number of antigens, including gluten.

Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique.

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.

Flat adenomas of the colon.

Twenty-nine flat adenomas of the colon from 18 patients were identified by histologic review of 340 surgically or colonoscopically removed adenomas from 210 patients. All lesions had a radial diameter of 1.0 cm or less. Twelve of 29 flat adenomas (41%) contained high-grade epithelial dysplasia, while only five of 127 polypoid tubular adenomas 1.0 cm in diameter or less (4%) contained high-grade epithelial dysplasia. Nine patients had multiple flat adenomas, and two patients had concurrent flat, ulcerated colonic carcinomas without an identifiable polypoid precursor adenoma. Colonoscopically and grossly, the lesions were described as sessile or flat, slightly raised plaques, which might be easily missed on colonoscopic examination. These findings suggest that flat adenomas may be a subtype of colonic adenomas with a propensity for development of high-grade epithelial dysplasia at a small size. These lesions may be precursors of small, flat, ulcerated colonic carcinomas. Heightened colonoscopic surveillance of patients in whom flat adenomas have been identified may be warranted.

Site-specific comparison of p53 immunostaining in squamous cell carcinomas.

The p53 tumor suppressor gene is believed to be the most commonly mutated gene in human cancer. p53 is thought to function as a negative regulator of the cell cycle, arresting cells in the G1 phase. This study examined the effects of different mutagenic environments on the incidence of p53 overexpression in squamous cell carcinomas (SCCs) from sun exposed and non-sun exposed squamous epithelium. An immunohistochemical analysis was undertaken in an attempt to assay SSC for p53 overexpression, an indirect measure of missense mutant p53. Positive nuclear staining for p53 was observed in 14 of 21 sun exposed SCCs, two of 19 vulvar/perianal SCCs, and 15 of 20 oral cavity SCCs. The number of positive anogenital tumors was low compared with that of both sun exposed (chi-squared, 1 df, P = .0004) and oral (chi-squared, 1 df, P < .0001) sites. It was concluded that p53 protein accumulation is common in sun-exposed cutaneous SCC and oral SCC compared with anogenital SCC, and thus it is hypothesized that the nature of the mutagenic environment in which SCC develops directly affects the incidence of immunohistochemically detectable p53-positive cells.

Quantitative image cytometry of infiltrating ductal carcinoma: comparison with prognostic parameters and reproducibility of histological grade.

Quantitative image cytometry was used to compare 18 parameters relating to ploidy, nuclear area, and chromatin texture to axillary lymph node status, tumor size, and histological grade for 34 infiltrating ductal carcinomas, each of which had been graded independently by each of six surgical pathologists. Zinc formalin-fixed, paraffinembedded tumors were assessed using the Elston and Ellis modification of the Bloom and Richardson histological grading scheme. When axillary lymph node-negative tumors were compared with those involving four or more nodes, % 2 c (diploid) cells, nuclear area, and eight of 12 chromatin texture parameters showed statistically significant differences. Carcinomas < 2 cm had more % 2 c (diploid) cells and fewer % > 4 c (hypertetraploid) cells than larger neoplasms. For tumors having nuclear pleomorphism score two versus those with score three, nuclear area, four of five parameters related to ploidy level, each of five parameters related to run-length matrix features and one of four co-occurrence matrix features showed significant differences. Nearly all of these cytometric parameters also showed significant differences for histological grade and mitotic count, which was strongly correlated with nuclear pleomorphism. In examining the cytometric parameters in relation to the interobserver reproducibility of histological grade and its components, the largest number of statistically significant parameters related to the nonreproducibility of nuclear pleomorphism. The findings indicate that as the grade of infiltrating ductal carcinomas increases, there are fewer % 2 c (diploid) cells and more % > 4 c (hypertetraploid) and % > or = 5 c (polyploid) cells. In addition, the cells of high grade tumors have larger nuclear areas and more small and large dense chromatin clumps, which increase in such number that they tend to join together. When compared with the cytometric parameters, nuclear pleomorphism is the most sensitive component of grade to nonreproducibility.

Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget's disease.

Formalin-fixed, paraffin-embedded tissue sections from 45 patients with mammary and extramammary Paget's disease were stained immunohistochemically with the use of a polyclonal antiserum directed against a 14-amino acid segment of the c-erbB-2 oncoprotein. Positive membrane staining, which correlates with gene amplification, was found in 15 of 19 cases (79%) of mammary Paget's disease, 4 of 13 cases (31%) of vulvar Paget's disease, none of 8 cases of scrotal Paget's disease, and none of 5 cases of perianal Paget's disease. Of the 19 patients with mammary Paget's disease, specimens of underlying breast tissue were available from 14; all contained a concurrent ductal adenocarcinoma. Concordance of c-erbB-2 antigen staining between the underlying breast carcinoma and the pagetoid component was observed in 12 cases. Of the 13 patients with vulvar Paget's disease, 2 had superficial stromal invasion, and 3 had underlying, deeply invasive adenocarcinomas. One superficially invasive case was positive for c-erbB-2 expression. One additional case of vulvar Paget's disease had an associated primary pagetoid endocervical adenocarcinoma that spread into the endometrium; both the endocervical and vulvar components stained positively for the c-erbB-2 antigen. The results of this study indicate that the c-erbB-2 oncoprotein may play a role in the pathogenesis of extramammary Paget's disease. These results also suggest that the c-erbB-2 oncoprotein may function in vivo to promote intraepithelial spread of adenocarcinoma cells.

Interobserver reproducibility of the Nottingham modification of the Bloom and Richardson histologic grading scheme for infiltrating ductal carcinoma.

The interobserver reproducibility of the Nottingham modification of the Bloom and Richardson histologic grading scheme for invasive breast carcinoma was tested. Six surgical pathologists from four institutions independently evaluated histologic grade and each of its three components for 75 infiltrating ductal carcinomas. The number of slides per case ranged from one to nine (median 3). Pairwise kappa values for agreement ranged from moderate to substantial (0.43-0.74) for histologic grade. Generalized kappa values indicated substantial agreement for tubule formation (0.64), moderate agreement for mitotic count (0.52), and near moderate agreement for nuclear pleomorphism (0.40). Normalizing the mitotic counts per mm2 showed only slight improvement in agreement over the published range of mitotic counts for three different field areas. The results suggest that steps to discriminate between categories for nuclear pleomorphism would likely be of benefit for improving the interobserver reproducibility of histologic grade. Nevertheless, the Nottingham modification of the Bloom and Richardson grading system is recommended as a suitable scheme for evaluating invasive breast carcinomas in the routine clinical setting.

Polyclonal carcinoembryonic antigen staining in the cytologic differential diagnosis of primary and metastatic hepatic malignancies.

In order to assess the utility of immunocytochemical staining of bile canaliculi with a polyclonal antiserum to carcinoembryonic antigen (pCEA) in the differentiation of primary hepatocellular carcinomas from metastatic malignancies, pCEA staining was performed on fine needle aspiration specimens from hepatic lesions in 60 patients. The original cytologic diagnoses were hepatocellular carcinoma in 22 patients, metastatic neoplasm or cholangiocarcinoma in 27 patients and benign hepatocytes in 11 cases. The cytologic diagnoses of malignancy were confirmed by surgical excision, autopsy or clinical investigations in 82% of the patients. Follow-up data, supported by pCEA staining, reversed the original cytologic diagnosis in three cases. Bile canalicular pCEA staining was identified in 18 of 22 cases of hepatocellular carcinoma and in all 11 benign hepatocellular aspirates. All 27 cases of metastatic malignancy or cholangiocarcinoma were negative for canalicular pCEA staining, although 11 cases exhibited cytoplasmic staining. Interpretation of pCEA staining was not affected by the intermingling of malignant cells and benign hepatocytes. Predictive values were 100% for a positive test and 87% for a negative test. These findings indicate that staining with pCEA antiserum is a useful adjunct in the differential cytologic diagnosis of malignant hepatic lesions.

In situ DNA hybridization of cervical small cell carcinoma and adenocarcinoma using biotin-labeled human Papillomavirus probes.

A preferential association of human Papillomavirus (HPV) type 18 with cervical small cell carcinoma and adenocarcinoma has been identified by in situ and blot hybridization analysis using radionucleotide-labeled DNA and RNA probes. We attempted to detect HPV DNA in nine cases each of invasive cervical small cell carcinoma and adenocarcinoma using biotin-labeled probes to HPV types 6/11, 16/31/33/35, and 18 with a peroxidase-conjugated streptavidin detection system. HPV type 18 DNA was detected within four of nine small cell carcinomas and one of nine adenocarcinomas. HPV types 16/31/33/35 were detected in one additional case of cervical adenocarcinoma. All HPV-positive small cell and glandular tumors showed a distinctive, punctate, often juxtanucleolar pattern of nuclear staining which involved the majority of carcinoma cells throughout each neoplasm. This pattern of HPV DNA labeling has not been observed in any of the HPV-positive typical squamous carcinomas or condylomas hybridized at our institution. It is possible that punctate nuclear HPV DNA staining is a marker of viral integration into the host cell genome. We conclude that in situ DNA hybridization with biotinylated probes, although less sensitive than detection of virally transcribed RNA, still allows detection of relatively low copy numbers of HPV DNA in cervical small cell carcinomas and adenocarcinomas. Furthermore, the spatial precision of biotinylated probes may provide morphological information not obtainable using radionucleotide-labeled probes.

Long-term soft-tissue effects of biliary extracorporeal shock waves: an animal study.

Although delayed effects of renal extracorporeal shock-wave lithotripsy (ESWL) have been reported, the long-term soft-tissue effects after biliary ESWL have not been investigated. We report soft-tissue effects seen up to 1 year after biliary extracorporeal shock waves were administered to 18 Yucatan pigs. The gallbladder received 4000 electromagnetic shock waves from a Siemens Lithostar overhead module. Blood samples were drawn from each pig for hematologic, coagulation, and biochemical profiles immediately before and then at prescribed time intervals after administration of shock waves. Autopsy and histopathologic examination of the gallbladder and surrounding organs were performed. Kidneys and adrenal glands also were examined in five pigs followed up for 1 year. There were no gross or microscopic abnormalities in 11 animals, including all five animals in the 1-year group in which kidneys and adrenal glands also were normal. One animal (3-week group) had two 2-mm foci of parenchymal necrosis in the right lobe of the liver, probably related to ischemia after shock-wave therapy. Transient rise in liver and pancreatic enzyme levels was seen in most animals after administration of shock waves. The levels returned to normal within 2 months in all but one animal. We conclude that biliary ESWL with the Lithostar Plus does not produce long-term histologic evidence of organ damage in Yucatan pigs.

Hairy leukoplakia after bone marrow transplantation.

Hairy leukoplakia in 10 patients after bone marrow transplantation was identified clinically and assessed histologically. In situ hybridization for Epstein-Barr virus and human papilloma virus confirmed Epstein-Barr virus in hairy leukoplakia in two cases, and human papillomavirus in three cases. All cases with clinical follow-up resolved without treatment. These findings suggest that severe immunosuppression after a bone marrow transplantation may result in the development of hairy leukoplakia, and that as the immunosuppression resolves after the transplant the lesions also resolve.

Oral manifestations of cytomegalovirus infection.

Disease caused by cytomegalovirus is reported with increasing frequency. Cytomegalovirus is an important pathogen in immunocompromised and immunosuppressed patients. The most common manifestation of cytomegalovirus infection of the gastrointestinal tract including the oral mucosa is ulceration. The role of cytomegalovirus in xerostomia, Sjögren's syndrome, and Kaposi's sarcoma is continuing to be investigated. This article reviews the oral manifestations of cytomegalovirus, including recently reported oral manifestations.