Heparin effect on DNA synthesis in a murine fibrosarcoma cell line: influence of anionic density.

The effects of heparin subfractions on DNA synthesis in a murine cutaneous fibrosarcoma cell line were examined. Porcine mucosal heparin was preparatively fractionated for anionic charge density by DEAE-Sephadex chromatography and for molecular weight by Sephadex G-100 filtration. The cell line was plated from confluent monolayer cultures and grown in medium and fetal bovine serum, with or without a heparin fraction at a final concentration of 10 micrograms/ml. At intervals thereafter, the cells were pulsed with [3H]thymidine. A low-charge density heparin fraction stimulated [3H]thymidine incorporation (cpm/mg protein and cpm/cell) during the first 3 days of growth compared to control values without added heparin, whereas a high-charge density heparin fraction had little of this effect (186 +/- 35% of control vs. 101 +/- 14%, respectively; P less than .05). The augmentation of DNA synthesis observed with the low-charge density fraction correlated with increased proportions of cells in S and G2 phases compared with those of the controls, as determined by flow cytofluorometry. Low- and high-molecular-weight heparin fractions did not significantly alter DNA synthesis. Heparin subfractions are thus heterogeneous with respect to their effect on cellular DNA synthesis in this tumor line.

Inheritance of nevus number and size in melanoma and dysplastic nevus syndrome kindreds.

Previous studies of the genetics of melanoma have focused on the dysplastic nevus syndrome (DNS). The variability in clinical and histopathological expression of affected individuals, however, has made definition and diagnosis of the syndrome difficult and subjective. Independent of the DNS, case-control studies have demonstrated the total number of nevi to be a significant risk factor for melanoma. In this article, we report results of genetic analyses of two quantitative nevus phenotypes that can be measured objectively in all subjects: the total number of nevi on an individual (TNN) and total nevus density (TND), a derived phenotype which incorporates both number and size of nevi. Ten kindreds ascertained for multiple cases of DNS-melanoma (multiplex ascertainment) and 16 kindreds and 19 solitary cases ascertained from a sequential list of melanoma cases without regard for family history (simplex ascertainment) were studied. Both phenotypes exhibited increased levels in relatives of probands compared with those in spouse controls. While neither TNN nor TND exhibited evidence for a major factor in the simplex pedigrees, a major factor was strongly indicated in the multiplex kindreds for TND. When both phenotypes were examined in more detail in the multiplex kindreds, the phenotype incorporating nevus size, TND, fit a mendelian pattern of inheritance better than the TNN. Significant residual familial correlations were found for both phenotypes. Parameter estimates from the best fitting genetic model indicated that a major gene may be responsible for 55% of the phenotypic variability of TND in the multiplex kindreds.

Influence of genes of the major histocompatibility complex on ulcerative dermal necrosis induced in mice by Mycoplasma arthritidis.

All mouse strains injected s.c. with Mycoplasma arthritidis developed severe abscesses in the subdermal tissues. However, M. arthritidis strain 14124 P10 also induced an ulcerative dermal coagulation necrosis in mouse strains expressing the k and d haplotypes but not in those expressing the b, q, or s haplotypes. The use of inbred and congenic mouse strains established that the ulcerative necrosis was associated with the haplotypes expressed at the H2 major histocompatibility complex (MHC). The gene restriction seen could be partially overcome by using a more virulent mouse-passaged strain of M. arthritidis (158 P10P9). The data suggest that genes of the MHC function by rendering certain mouse strains more susceptible to an as yet unidentified necrotizing moiety. The close histologic resemblance of the dermal necrosis induced by M. arthritidis to certain human diseases such as necrotizing fasciitis, the ulcerative lesions induced by Mycobacterium ulcerans, and the crepitant and gangrenous cellulitides may therefore provide a unique model to study the genetic factors and mechanisms of pathogenesis in these latter human conditions.

Infective endocarditis associated with cell wall-deficient bacteria. Electron microscopic findings in four cases.

Although cell wall-deficient bacteria have been isolated in vitro from cases of endocarditis, no pathogenic role has been established for these forms in human disease. One criterion difficult to satisfy is the demonstration of these variants in human tissue, and electron microscopic documentation has not been reported. Cardiac valvular vegetations from four cases of endocarditis were examined by electron microscopy because of unusual histologic features of minimal inflammation and organization and small organisms that stained poorly by Gram stain. Although cell wall-complete bacteria were identified in the specimens, each showed the presence of cell wall-deficient forms within the vegetations; these variants predominated in three cases. Since manifestations of infective endocarditis were present in three cases and conventional cultures were negative, the evidence indirectly suggests a pathogenic role for these aberrant bacteria in human disease.

Tumor vascularity, proliferation, and apoptosis in human melanoma micrometastases and macrometastases.

BACKGROUND: Clinically undetectable or dormant metastases (micrometastases) probably account for disease recurrence, ie, clinically evident metastases, in patients after disease-free intervals of variable length. Recently developed animal models have shown that dormancy may potentially be explained by the fact that these micrometastases are not vascularized and have comparable rates of cellular proliferation and programmed cell death (apoptosis), enabling them to remain viable indefinitely but not to show progressive growth. OBSERVATION: We report for the first time that melanoma micrometastases from humans are similarly not vascularized (mean number of microvessels, 10.2), have significantly lower rates of tumor cell proliferation (mean, 2.4%), comparable rates of proliferation and apoptosis (means, 2.4.% and 0.2%, respectively), compared with melanoma macrometastases, which have significantly greater tumor vascularity (mean number of microvessels, 18.7), higher rates of proliferation (mean, 18%), and higher rates of proliferation relative to apoptosis (means, 18% vs 1.6%). Tumor vascularity was quantified using the lectin Ulex europaeus agglutinin I to identify the number of microvessels per unit area (microscope ocular grid with an area of 7.84 x 10(-2) mm2 at x400 magnification). Melanoma cell proliferation rate was assessed with the MIB-1 antibody (Ki-67) as the number of positive nuclei per total number of tumor nuclei counted at x400 magnification. Apoptosis was quantified using the method of terminal deoxynucleotidyl transferase-medicated deoxyuridine triphosphate-biotin nick end labeling. The number of positive nuclei were quantified per total number of tumor nuclei; usually 200 tumor nuclei were counted at x400 magnification. CONCLUSION: We report, for the first time, that human micrometastases demonstrate attributes, ie, the lack of significant tumor vascularity and low but comparable rates of proliferation and apoptosis, that may explain the dormant state.

Fatty metamorphosis of the liver associated with jejunoileal bypass. Report of five cases.

Detailed postmortem examination was carried out on five patients who died three months to four years after jejunoileal bypass for obesity. A spectrum of histological changes was observed in the liver, with pericentral fat deposition being a common feature. Evidence of previous and/or ongoing liver cell dropout with accompanying polymorphonuclear and mononuclear infiltration was seen in all cases, but Mallory hyalin was not detected. Liver function abnormalities included decreased plasma protein levels, decreased prothrombin activity, increased serum alkaline phosphatase levels, and variable elevations of the serum transaminases, bilirubin, and ammonia concentrations. The pattern of the hepatic disease does not resemble protein deficiency. An uncharacterized hepatotoxin or toxic effect of hepatic fat accumulation may play a significant role in the changes observed in these patients.

Immunologic studies documenting the development of mycosis fungoides following successful therapy of a large-cell lymphoma.

We describe a patient who developed mycosis fungoides, a T-cell neoplasm, two years following the chemotherapy and radiotherapy of a diffuse, large-cell ("histiocytic") lymphoma. Immunologic studies documented a "null-cell" phenotype for the large-cell lymphoma. Therefore, the proliferation of two distinct malignant lymphoid clones is believed to have developed in the patient. The value of immunologic marker studies in this case was to aid in the diagnosis of the two separate neoplasms, as well as to aid in the selection of appropriate specific therapy directed against each neoplasm. As the association of large-cell lymphoma and mycosis fungoides in one patient represents a very rare event, we discuss several hypotheses about the possible interrelation of the two neoplasms.

Dysplastic naevi with moderate to severe histological dysplasia: a risk factor for melanoma.

BACKGROUND: The risk of malignant melanoma associated with histologically dysplastic naevi (HDN) has not been defined. While clinically atypical naevi appear to confer an independent risk of melanoma, no study has evaluated the extent to which HDN are predictive of melanoma. OBJECTIVES: To estimate the risk of melanoma associated with HDN. Secondarily, the risk associated with number of naevi and large naevi is estimated. METHODS: We enrolled 80 patients with newly diagnosed melanoma along with 80 spousal controls. After obtaining information on melanoma risk factors and performing a complete cutaneous examination, the most clinically atypical naevus was biopsied in both cases and controls. Histological dysplasia was then assessed independently by 13 dermatopathologists (0, no dysplasia; 1, mild dysplasia; 2, moderate dysplasia; 3, severe dysplasia). The dermatopathologists were blinded as to whether the naevi were from melanoma subjects or controls. Multivariate analyses were performed to determine if there was an independent association between the degree of histological dysplasia in naevi and a personal history of melanoma. RESULTS: In persons with naevi receiving an average score of > 1 (i.e. naevi considered to have greater than mild histological dysplasia), there was an increased risk of melanoma [odds ratio (OR) 2.60, 95% confidence interval (CI) 0.99-6.86] which persisted after adjustment for confounders (OR 3.99, 95% CI 1.02-15.71). Very few dermatopathologists reliably graded naevi of subjects with melanoma as being more dysplastic than naevi of control subjects. Among the entire group, the interobserver reliability associated with grading histological dysplasia in naevi was poor (weighted kappa 0.28). CONCLUSIONS: HDN do appear to confer an independent risk of melanoma. However, this result may add more to our biological understanding of melanoma risk than to clinical assessment of risk, because HDN assessed by a single pathologist generally cannot be used to assess risk of melanoma. Future studies should be directed at establishing reproducible, predictive criteria for grading naevi.

Dansyl (5-dimethylaminonaphthalene-1-sulphonyl)-heparin binds antithrombin III and platelet factor 4 at separate sites.

Antithrombin III binds to, and thereby augments the fluorescence of, dansyl-(5-dimethylaminonaphthalene-1-sulphonyl)-heparin; platelet factor 4 binding to the fluorescent heparin has little of this effect. Competition studies in which antithrombin III competes with platelet factor 4 for heparin binding demonstrate that heparin can simultaneously bind both proteins.

Genetic basis of susceptibility to melanoma.

The search for candidate genes involved in the genesis of common cancers has traditionally been hampered by ambiguities in the process of determining by reliable, clinical criteria which persons harbor the genetic lesion that confers malignant susceptibility. In the case of cutaneous melanoma, the existence of genetic susceptibility has long been evident from its tendency to cluster in families, but it has been unclear until recently whether the genetic basis of familial melanoma derives from the concerted interaction of multiple genes or from a major locus with properties of a tumor suppressor gene. The original strategy used to circumvent difficulties in identifying those who harbor the genetic defect exploited a proposed melanoma precursor lesion, the dysplastic nevus, as the phenotypic marker from which the presence of the melanoma-associated genotype was inferred. That strategy in genetic linkage studies provided the first indication of a major gene for melanoma and assigned the locus to the short arm of chromosome 1. In part because the criteria for the dysplastic nevus have been neither well-defined nor generally agreed upon, multiple independent attempts to confirm the assignment of a gene to that location have failed. The probable map position of a major gene became clear when the most frequently deleted region of the human genome in melanoma tumors was localized to chromosome 9p. The significance of this assignment was established when genetic linkage studies of multiple melanoma kindreds subsequently evaluated the correlated inheritance between melanoma gene carriers, as assigned by a history of melanoma, and molecular markers for DNA polymorphisms near the 9p candidate region; this analysis provided strong statistical evidence of linkage to a melanoma susceptibility locus. Once this candidate tumor suppressor gene) as well as other relevant suppressor loci that may exist is actually cloned and characterized, rapid advances can be expected in our understanding of the pathophysiologic basis for development of melanoma. This will provide opportunities for exploring the mechanisms underlying defects in the gene and the molecular consequences of its loss of function. It will then be possible to identify precisely those persons with a genetic risk for melanoma; as a result, surveillance efforts can be more appropriately focused than has heretofore been possible.

A perspective on the dysplastic nevus controversy.

In 1992 an impartial scientific panel concluded that the term "dysplastic nevus" has created significant confusion and thus has outlived its usefulness. This chapter reviews the evolution of the controversy and offers a basis for understanding the panel's recommendations.

Comparable efficacy of 2% minoxidil gel and solution formulations in the treatment of male pattern alopecia.

Sixty-four healthy men with early male pattern baldness completed a 6-month, double-blind, placebo-controlled study in which the efficacy of 2% minoxidil gel and solution formulations was compared. Treatment with topical 2% minoxidil in both formulations resulted in a statistically significant increase in terminal hair growth referenced against baseline counts. The patients' subjective assessments of hair growth did not correlate well with actual increase in terminal hair counts.

Glycosaminoglycans and the substrate attachment of murine myeloma, 3T3, and cutaneous fibrosarcoma cells.

The attachment of murine myeloma, 3T3, and cutaneous fibrosarcoma cells to substrates of either fibronectin, type I collagen, or two types of tissue culture plastic was examined in the presence and absence of specific exogenous glycosaminoglycans. Fibrosarcoma and 3T3 cells were found to be nondiscriminatory with respect to their avidity of attachment to substrates of either of the proteins or of conventional tissue culture plastic, whereas the myeloma cells attached significantly less well to a substrate of collagen than to the other two matrices. On tissue culture plastic and collagen the fibrosarcoma cells attached more rapidly than did the other two cell types. Selective and partial inhibition of cell attachment to type I collagen, and, to a lesser extent, fibronectin, occurred upon preincubating these substrates with the sulfated glycosaminoglycans, heparin and heparan sulfate, at concentrations of 1 to 100 micrograms/ml; for 3T3 cells heparin was significantly more inhibitory (mean maximal inhibition of approximately 40%) than were two heparan sulfate fractions. Attachment of fibrosarcoma and 3T3 cells to a nitrogenated tissue culture plastic surface with a net positive charge (Primaria) was nearly 50% inhibited by heparin at the higher concentration and to a lesser extent by the two heparin sulfate fractions. Myeloma cell attachment to this same substrate was inhibited, to a lesser degree, by all three sulfated glycosaminoglycans. Hyaluronic acid, dermatan sulfate, and chondroitin 6-sulfate were inactive in our attachment assays. We suggest that the functional role of glycosaminoglycans in substrate attachment may vary depending on the cell type and the matrix involved in the specific interaction. In particular, the net charge of the substrate appears to be an important factor, and on positively charged surfaces these substances may serve a greater function. However, since nearly complete abrogation of cell attachment should have been achievable by some of the exogenous preparations if cell surface sulfated glycosaminoglycans were to comprise the major cellular binding sites for matrices, we conclude that it is unlikely that these complex polysaccharides function as the principal determinant of simple cell attachment.

Reliability of skin biopsy pathology.

BACKGROUND: The diagnosis of skin disease by histologic examination is regarded as the reference standard upon which therapy and follow-up are determined. Our study investigated the reliability of skin biopsy diagnosis requested by family physicians and physicians' assistants. METHODS: Biopsy diagnoses by a community-based pathology group were reinterpreted by our study dermatopathologist on a sample of 119 skin biopsies randomly selected from the 1844 biopsies performed by family physicians and physicians' assistants at a large Washington State health maintenance organization during a 4 1/2-year period. RESULTS: There were 107 exact matches and 3 mismatches of premalignant lesions and 6 mismatches of benign diagnoses. In addition, two melanomas diagnosed by the community-based pathologists were interpreted as benign by our study dermatopathologist. A third melanoma diagnosed by the community-based group was interpreted as a poorly differentiated squamous cell cancer by the university dermatopathologist. The weighted kappa, 0.83, indicated excellent interrater agreement. CONCLUSION: Although our study showed excellent interrater concordance of skin biopsy interpretation, there was disagreement about three melanomas between a community-based general pathology group and our study dermatopathologist. The melanoma disagreement is consistent with previous studies that found poor interrater agreement for early melanomas. The community-based pathologists were uncertain about two of these melanomas, and as part of their quality control and review procedures requested confirmation by an expert pathologist, who agreed with the melanoma diagnosis. Family physicians are justified in requesting a second opinion (if not automatically requested by a community laboratory) when the histopathologic diagnosis is not in concordance with the clinical history or impression or when the pathologist is unsure of the diagnosis.

Persistent melanocytic lesions associated with cosmetic tanning bed use: "sunbed lentigines".

A patient with persistent melanocytic lesions after tanning bed use is described. A review of the literature provides two additional examples of similar clinical and histologic presentations after UVA exposure without psoralen. To our knowledge, this is the first reported case of "sunbed lentigines" in the United States.

Modified erythrocyte uroporphyrinogen I synthase assay, and its clinical interpretation.

Assay of erythrocyte uroporphyrinogen I synthase is an accepted diagnostic test for acute intermittent porphyria, particularly in those individuals who are asymptomatic or in whom the disease is not biochemically manifested by excretion of excess porphyrin precursor. The assay described is based upon a coupled-enzyme procedure in which added delta-aminolevulinic acid and its dehydratase present in erythrocytes are used to generate porphobilinogen as substrate for uroporphyrinogen synthase. Zinc and dithiothreitol are added with preincubation to give maximum activity and reproducibility. These agents also prevent inhibition by lead. Healthy young women had a mean activity of 40 nmol of porphyrin formed per milliliter of erythrocytes per hour, men and activity of 38 nmol/ml/h. Preparation of control specimens is described. This assay gave within-day CVs ranging from 1.9 to 2.8%. Precautions in interpretation of results are discussed.

A kindred with congenital vellus hair cysts.

Vellus hair cysts were present from birth in two of four siblings and their mother. There has been no tendency for remission. This is the first report of vellus hair cysts appearing at birth, and the second report of a kindred in which there was apparent autosomal dominant inheritance. This disorder appears to represent a developmental anomaly of vellus hair follicles.