Illness behavior in children of chronic pain patients. Recent studies on pain behavior have pointed to a relationship between expression of illness in patients with chronic pain and reports of physical complaints in other family members. This article includes two related studies. The aim of the first study was to determine whether, among pain patient families, parents who exhibited higher levels of illness behavior and emotional distress were more likely to report that their children had frequent pain complaints. The aim of the second study was to assess whether children of patients with chronic pain were perceived by their parents to have more pain and illness behavior than children of non-pain parents. In the first study, forty-two primary caregivers in families with a parent with chronic pain completed questionnaires regarding their children's pain and illness behavior. In the second study, report of somatic complaints in children of chronic pain parents was compared to complaints in fifty-five children of parents without chronic pain. Results of the first study showed that frequently reported pain in the child was associated with significantly higher levels of parent disability, pain behavior and emotional distress. Results of the second study showed that children of patients with chronic pain were reported to have more frequent abdominal pain and to use more medication than children of parents without pain. The findings of these studies suggest that children of parents with chronic pain may be at risk for illness behavior, especially when the parents exhibit emotional reactions to their pain.


Pseudomonas vesicularis bacteraemia.

Pseudomonas vesicularis bacteraemia. A case of repeated episodes of Pseudomonas vesicularis bacteraemia, in a 54-year-old woman with a past history including systemic lupus erythematosus and chronic active autoimmune hepatitis is reported. She was treated with tobramycin and ceftazidime but bacteraemia persisted until surgical resection of the infected tissue was performed.


Munchausen syndrome by proxy.

Munchausen syndrome by proxy. This study examined the indicators of possible presence of Munchausen Syndrome by Proxy (MSBP) in pediatric inpatients discharged against-medical-advice (AMA), in cases transferred to another hospital, and in regular discharges. In a two-year period, there were fourteen AMA discharges and twenty-four transfer cases. These were compared to a randomly selected sample of forty-one regular discharges occurring during the same time period, matched for age, sex and pediatric service. The records were scored for the presence or absence of six MSBP characteristics, which had been developed from a review of MSBP literature. The findings indicated a suspicion for MSBP in 64 percent of the AMA cases, 8 percent of the transfer cases, and none in regular discharges. The findings suggest that AMA discharges need to be examined for the possible presence of MSBP.


Paramenstrual aggression in mentally retarded adult ameliorated by buspirone. This case examined the efficacy of buspirone therapy as a reductive technique for severe treatment resistant paramenstrual aggression. Aggressive outbursts of an institutionalized mentally retarded adult were analyzed and correlated with menstrual cycling. Behavior was closely monitored, and after ineffective trials of antipsychotics, benzodiazepines and beta blockers, a trial of buspirone was initiated. Buspirone therapy resulted in a marked decrease in aggressive incidences, and substantial increases in desirable behaviors. As a result of the subjects improvement, doses of concurrent psychotropic medications were reduced or discontinued altogether. The authors conclude that buspirone is an effective pharmacotherapy for paramenstrual aggression in the developmentally disabled, and encourage similar experimentation when behavior seems to be otherwise untreatable.


A review of the postconcussion syndrome. This review will focus on aspects of the postconcussion syndrome (PCS), including accompanying symptomatology, neuropsychological changes, brain imaging studies and treatment. In each topic area, those research studies resulting in the most interpretable data are reported. Since there is little research in some aspects of the PCS, some studies of limited merit are described, with their limitations outlined, in lieu of not reporting any study. The section on psychopharmacology largely consists of opinions of recognized clinicians, since there is almost no research on the psychopharmacology of PCS. Mild traumatic brain injury is a relatively frequent occurrence which often results in the postconcussion syndrome (PCS), consisting of complaints of irritability, fatigue, headache, difficulty concentrating, dizziness, and memory problems. Anxiety and depression are also frequently present, especially later in its course. Although the PCS has often been thought to reflect a psychological response to injury, there is considerable recent evidence to suggest that it is primarily a physiologic disturbance. For most individuals, treatment consists primarily of education of the patient and his/her family, along with supportive counseling regarding emerging problems at work or at home. A subgroup of patients, however, may require psychopharmacologic intervention. More research is needed in all aspects of PCS, especially its neurophysiology and pharmacologic treatment. Relationships between neurophysiological changes and behavioral and neuropsychological changes are unknown. New imaging techniques, such as single-photon emission tomography, and positron emission tomography will likely play an important role in understanding the physiology of this disorder.


Monoclonal antibodies recognising pol and env proteins of mammalian retroviruses. Mouse monoclonal antibodies were generated for three 20- to 23-mer synthetic peptides whose amino acid sequences were highly conserved in the pol or env regions of 14 mammalian and two avian retroviruses. By Western-blot analysis, these antibodies recognised the epitopes in major and minor pol or env proteins of six mammalian type-C and -D retroviruses. One of the anti-env monoclonal antibodies was found to recognise all the env transmembrane proteins of six mammalian retroviruses examined.


Transmission studies with simian immunodeficiency virus of macaques; persistent infection of baboons. The host range of SIVmac was investigated in three monkey species. Blood-borne and cell-adapted virus inocula obtained from a rhesus macaque infected with SIVmac251 were compared. African green monkeys were not susceptible to infection, whereas baboons and rhesus macaques became persistently infected and showed similar patterns of seroconversion. However, in contrast to the macaques, no clinical or histopathological evidence of disease was seen in the baboons 2 years after virus inoculation. Thus baboons could be used as an alternative to macaques in vaccine development studies with this particular isolate of SIVmac. Furthermore, this system may be useful for the investigation of factors responsible for disease progression.


Experience with subfascial ligation for varicose veins in Tanzania: a brief communication. From January 1987 through December 1989, ten patients with varicose veins complicated by postphlebitic ulcers were treated at Muhimbili Medical Center, Tanzania, by subfascial ligation of perforating veins. The technique employed was a posterior subfascial approach which avoids making the skin incision through the ulcer itself. The follow up periods ranged from one to three years and there was no recurrence of the ulcer in any of the patients in the study. The major indication for performing the procedure was the presence of a varicose stasis ulcer. Before surgery, all patients had a complete peripheral vascular examination to exclude deep venous thrombosis. Healing of varicose ulcers and elimination of stasis eczema had to be achieved four weeks before surgery. The authors maintain that patients who present with varicose veins of the lower leg and a postphlebitic stasis ulceration invariably have incompetence of the valves in the perforating veins and should be treated by subfascial ligation of the perforating veins.


Estimation of the urinary galactitol level in children by capillary gas chromatography. A rapid and efficient capillary gas chromatography was utilized to measure urinary galactitol in 133 non-galactosemic children on a free diet. The children aged from 12 days to 14 years were divided by age into six groups. The urinary galactitol concentration was found to be the highest (64.04 mmol/mol creatinine) in the neonatal group and the lowest (7.12 mmol/mol creatinine) in the group over 2 years old. It is concluded that the urinary concentration of galactitol is strongly age-dependent.


Helicobacter pylori infection and gastroduodenal disease: a comparison of endoscopic findings, histology, and urease test data. To determine the prevalence and significance of Helicobacter pylori (H. pylori) infection, biopsies of the antral mucosa were obtained from 139 patients and 43 asymptomatic volunteers. The specimens were examined by hematoxylin-eosin staining and the ureas test. The detection rate of H. pylori by histologic examination was 91.3% in patients with duodenal ulcer, 75.0% in those with combined duodenal and gastric ulcer, 63.6% in those with gastric ulcer, 22.9% in those with gastric carcinoma, 36.4% in those with gastric adenoma, 14.3% in those with gastric hyperplastic polyp, and 51.7% in those with gastritis, and the respective percentages detected by the urease test were 91.3%, 75.0%, 54.5%, 28.6%, 27.3%, 14.3%, and 44.8%. H. pylori was also detected in 10/43 (23.3%) asymptomatic healthy volunteers by histology and the urease test. The prevalence of H. pylori was significantly higher in the patients than in the asymptomatic healthy volunteers (p < 0.05). H. pylori was detected in 62.9% of patients with endoscopic erosive gastritis and in 97.9% of those with histologically proven chronic active gastritis. The urease test was positive in 77/82 patients who were histologically positive for the organism (sensitivity: 93.9%), and it was negative in 98/100 patients who were negative by histology (specificity: 98.0%). Thus, there was over 90% agreement between the urease test and histology. Our investigations showed that H. pylori was closely related to peptic ulcers and antral gastritis, and that the urease test provides a simple, rapid and accurate diagnosis of H. pylori infection.


Cellular aging and expression of fibronectin. Several changes in the functional characteristics of fibronectin (FN) have been noted as cells become senescent in culture. In a previous report we showed that the steady state level of FN mRNA increases significantly during the process of in vitro cellular aging in a fibroblast strain. Because a phenomenon observed in one cell strain may not be the case in other cell strains, we extended the previous study and confirmed that this is a common phenomenon in at least two fibroblast strains of different origin. The greatest change in the proportion of cells expressing high levels of FN occurs near the end of a culture's proliferative potential. The proportion of cells unable to synthesize DNA follows a similar pattern. We also found that increasing cell size correlates closely with higher levels of FN expression. Thus, there is a clear correlation between increased FN mRNA content and in vitro cellular senescence. In order to see if this phenomenon could also be observed in cells aged in vivo, we analyzed cells aged in vivo. We found that fibroblasts from donors of higher age show lower labeling index, express a higher level of FN and come to have a larger cell area, similar to cells aged in vitro. This strongly suggests that a fibroblast in vivo ages with aging of the individual in like manner to that observed in in vitro aging cells, that is, by exhausting division potential. This supports studies using in vitro aging cells as a model for cellular aging in vivo.


Light and electron microscopic observation in a case of congenital renal proximal tubular dysfunction. We report a case of congenital renal proximal tubular dysfunction (CRPTD) accompanied by IgA nephropathy. The mesangial matrix was slightly increased with depositions of IgA and C3. Podocytes contained many clear vacuoles. Cuboidal cells, as well as squamous cells, lined the parietal epithelium of Bowman's capsule, although the functional or pathological significance of the cuboidal cells is unknown. The proximal tubular cells appeared to have a less-developed apical endocytic complex, basal infoldings and fewer lysosomes. These morphological changes may reflect proximal tubular dysfunction.


Susceptibility of methicillin-resistant Staphylococcus aureus clinical isolates to various antimicrobial agents. II. Isolation of arbekacin-resistant strain. Resistance patterns against 24 antimicrobial agents were examined for 50 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated at Hiroshima University Hospital during the period October 1990 and July 1991. Overall resistance (the percentage of highly and moderately resistant strains) to the antimicrobial agents is summarized as follows: methicillin 100%, flomoxef 100% (beta-lactams); kanamycin 94%, tobramycin 94%, amikacin 100%, isepamicin 94%, gentamicin 80%, dibekacin 94%, arbekacin 2% (basic oligosaccharide group/aminocyclitols); ofloxacin 96%, temafloxacin 96%, levofloxacin 96% (fluoroquinolones); erythromycin 98%, clarithromycin 98%, josamycin 30% (macrolides); vancomycin 0% (glycopeptide); tetracycline 94%, minocycline 94% (tetracyclines); fosfomycin 100%; mikamycin B 30%, nosiheptide 0% (peptide); rifampicin 2% (ansamycin); streptomycin 2% (basic oligosaccharide group); chloramphenicol 2%. Arbekacin resistance was observed in one case: the cross resistance was complete among the aminocyclitol antibiotics tested in this study and streptomycin, probably due to the ribosomal alteration.


Elastosis in breast--correlation with epithelial proliferation in benign disease and carcinomatous growth. Elastosis in the breast is an unusual phenomenon and its morphogenesis has not yet been fully ascertained. The degree of elastosis in the breast associated with benign diseases, including fibroadenoma and fibrocystic disease as well as with breast carcinoma, was examined with special reference to the correlation between the degree of epithelial proliferation and elastosis. Using the immunohistochemical method, the presence of elastase (EL) and alpha 1-antichymotrypsin (ACT), one of the protease inhibitors, in these epithelial cells was also investigated to elucidate the role of an imbalance in these enzymes in the morphogenesis of the elastosis. Consequently, it was shown that there is a tendency in fibroadenoma and fibrocystic disease, for epithelial proliferation to be related to the degree of elastosis, and that the lack of EL in proliferated epithelial cells might play a role in the occurrence of elastosis, although ACT has no significant correlation. On the contrary, in our study noninvasive carcinoma showed marked periductal elastosis but no stromal elastosis, while invasive carcinoma showed various degrees of periductal and stromal elastosis. In invasive carcinoma, especially scirrhous carcinoma, the degree of ACT in cancer cells correlated well with stromal elastosis, although there was no correlation with EL. These findings suggest that an imbalance of the protease-antiprotease system, produced by epithelial cells of the breast, contribute to the morphogenesis of elastosis, although the physiological event, aging, is only marginally related to elastosis. Further investigation of the cells producing elastin and regulatory factors may be necessary.


Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis. Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.


Expression of intestinal alkaline phosphatase in human organs. Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.


Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera, Culicidae). The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of sperm-oocyte interaction.


Protein kinase C (alpha, beta, gamma) in Pacinian corpuscle. Immunocytochemical demonstration of protein kinase C (PKC) subspecies (alpha, beta, gamma) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC alpha-immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive alpha-IR. Very weak PKC beta-IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC beta-IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC gamma-IR was not detected in any part of the corpuscle. The strong PKC alpha-IR in the inner core and the presence or absence of PKC alpha-, beta-, and gamma-IR in the axon terminal are discussed from the point of view of the functional aspects of each part.


Glycosaminoglycans and fibrillar collagen in Priapulida: a histo- and cytochemical study. The distribution of glycosaminoglycans and fibrillar collagen was studied in various tissues of priapulids, which represent an ancient group of marine metazoa. Sulphated glycosaminoglycans, as demonstrated at the electron microscopical level by Cupromeronic blue, were predominantly found in the cuticle, in basement membranes and also in the narrow connective tissue space below epidermis and anterior intestine. On the basis of their morphology the Cupromeronic blue precipitates could be divided into several groups. Fibrillar collagen occurred in the connective tissue under the epidermis and the epithelium of the anterior intestine. The spatial interrelationship between fibrillar collagen and glycosaminoglycans lacked with some exceptions, the high regularity found in connective tissues of other invertebrates and of vertebrates. This might be related to the special skeletal system of priapulids, consisting mainly of a strong extracellular cuticle and the turgor of the fluid-filled body cavity. In such a system the usual supportive structures seem to be of less functional significance.


Use of a murine T-cell hybridoma expressing human T-cell receptor alpha- and beta-gene products as a tool for the production of human T-cell receptor-specific monoclonal antibodies. We describe the production of mouse monoclonal antibodies specific for the human TcR using as the immunogen transfected murine T-cell hybridoma cells coexpressing mouse CD3 with human Jurkat TcR alpha and beta chains. The shortage of monoclonal antibodies (mAbs) specific for the human TcR-V alpha and V beta families reflects the difficulties in their production by conventional methods using whole human T cells or purified soluble receptors as immunogens. As an alternative strategy to circumvent these difficulties, we have generated a transfected mouse T-cell line expressing a human (Jurkat) TcR alpha beta dimer in a complex with mouse CD3. The parental mouse T-cell line, TG40, is a cell surface TcR-negative, cytoplasmic CD3-positive variant of the mouse T-cell hybridoma 2B4. The human-TcR alpha beta expressing mouse transfectant was used to immunize mice with the same genetic background as the parent mouse T-cell line, and a human TcR-specific response was successfully achieved. MAb-producing hybridomas were generated by fusing spleen cells from the immunized mice with the mouse myeloma cell line NSO. Of 124 hybridoma supernatants screens, 72 showed reactivity to the human T-cell line Jurkat. Twenty-four of the hybridomas producing human (Jurkat) TcR-specific antibodies were cloned and screened for reactivity to Jurkat TcR. Several IgG2b and IgM mAbs specific for the Jurkat T cell line were selected on the basis of their ability to modulate surface CD3 expression on Jurkat cells. Most of the antibodies do not stain other TcR-expressing human T cell leukemia cell lines, implying specificity for the variable domains of the Jurkat TcR.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of an automated method of percent reactive antibody determination. A fluorescence-based automated method of percent reactive antibody (PRA) analysis is described. This method utilizes the conventional antibody-mediated, C'-dependent lymphocyte microcytotoxicity assay to detect alloantibodies, but replaces the eosin-based method for detection of cell death with a fluorescence-based method. To identify viable cells, lymphocytes were pretreated with carboxy fluorescein diacetate (CFDA), which fluoresces green, to identify viable cells. To identify dead cells after the reaction with antibody and C', they were treated with propidium iodide (PI), which fluoresces red. Pretreatment of lymphocytes with CFDA did not affect their ability or interact with alloantibodies in the microcytotoxicity assays. When visually analyzed, detection of cell death by fluorescence was as sensitive as detection by eosin exclusion. However electronic detection of fluorescence was slightly more sensitive than visual detection. Automation of the fluorescent method required a calculation that converts electronic data to an ASHI score for cell death. One such method is described and evaluated. Both the automated and the conventional methods of analysis were used to obtain PRA values for various sera. There was good correlation between the PRA values obtained with the automated method versus the conventional method. Further, there was good correlation for PRA-derived alloantibody specificities obtained with the automated method versus the conventional method. These data demonstrate that automated fluorescence-based PRA analysis is an effective and practical alternative to conventional PRA analysis.


HLA-DR and -DQ genotypes of celiac disease patients serologically typed to be non-DR3 or non-DR5/7. The susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular HLA-DQ alpha/beta heterodimer encoded by the DQA1*0501 and DQB1*0201 alleles, in cis position on the DR3-DQ2 haplotype or in trans position by DR5-DQ7/DR7-DQ2 heterozygotes. However, exceptional patients exist who are neither DR3 nor DR5/DR7, particularly among Southern European populations. We therefore examined the DRB1, DQA1, and DQB1 alleles of 13 Spanish CD patients who were serologically typed to be neither DR3 nor DR5/DR7. Five patients were found to carry the DQA1*0501 and DQB1*0201 alleles either in cis or in trans position, three of them had previously been serologically mistyped. However, two of these patients carried DQA1*0501 and DQB1*0201 on haplotypes other than DR3 or DR5 in combination with DR7. One of the latter patients carried an unusual DR4-DQ2 haplotype, while another had an unusual DR8-DQ2 haplotype. Four of the remaining eight patients carried DR4-DQ8 haplotypes. Taken together, our findings provide further evidence that the DQ alpha/beta heterodimer encoded by the DQA1*0501 and the DQB1*0201 alleles confers the primary HLA-associated susceptibility to develop CD. However, our studies also corroborate that a second (and "weaker") HLA-associated CD susceptibility gene may be present on some DR4-carrying haplotypes.


HLA and immunoglobulin polymorphisms in idiopathic dilated cardiomyopathy. Dilated cardiomyopathy (DCM) is an idiopathic heart muscle disorder. The presence of circulating cardiac antibodies and the association with HLA-DR4 are consistent with autoimmune pathogenesis in a subset of patients. Sixty-eight DCM patients and 277 controls were typed for IgG heavy-chain constant region (Gm) and kappa light-chain (Km) allotypes. All patients and 210 of the 277 controls were HLA-DR typed. The Gm (1, 3, 17; 23; 5*, 21, 28) phenotype was overrepresented in DCM compared with controls (25% vs 13%, p = 0.0139, pc = NS, RR = 2.23). The frequency of this phenotype was higher in patients with younger age at onset, shorter symptom duration, and among those who were positive for cardiac as well as for non-organ-specific autoantibodies than in controls. A higher frequency of the Gm (1, +/- 2, 3, 17; +/- 23; 5*, 21, 28) heterozygous phenotypes was also found in DCM compared to controls (40.91% vs 26.89%; p = 0.02, pc = 0.04, RR = 1.88). The finding of Gm heterozygosity in DCM was associated with serum positivity for cardiac antibodies. A higher proportion of DCM patients were positive for both the Gm (1, 3, 17; 23; 5*, 21, 28) phenotype and HLA-DR4 compared to normals (3/68 vs 0/210; p = 0.04, RR = 22.50).(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative studies of isolated CD3: CD8, CD3: CD3, and monovalent CD3 binding on CD8+ T-cell activation: model of progressive T-cell receptor aggregation synergism. The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.


HLA polymorphism in a Mataco South American Indian tribe: serology of class I and II antigens. Molecular analysis of class II polymorphic variants. In the present study, HLA-A, B, C, DR, DQ, and DP loci were analyzed in a group of Mataco Amerindians of Argentina. Using reagents from the 11th International Histocompatibility Workshop (11th IHW), class I specifities such as Bw70, Bw75, and Bw48 were found in this population, other than the HLA determinants commonly described in South American Indians. The class II antigens found were DR4, DRw14, and DRw8 at the DR locus, and DQw4 and DQw7 at the DQ locus. The analysis of DRB1-DR4 related alleles, performed by PCR amplification and oligonucleotide probe hybridization, showed the presence of DRB1*0403, *0404, *0405, and *0411 in individuals from this ethnic group. By the analysis of DRB1-DRw14 related alleles, two variants were found: DRB1*1402 and DRB1*1406, the latter provisionally called DRB1 14.6 in 11th IHW. The DRw8-related allele present was DRB1*0802. The analysis of DRB3 gene revealed only the presence of DRB3*0101 allele in DRw14 individuals. DPB1 locus was also analyzed in unrelated individuals of the same population. Only five DPB1 alleles were found: DPB1*0201, *0301, *0402, *0501, and *1301 over the 19 previously described in the literature. These findings emphasize the restricted HLA class I and II variation observed in this ethnic group as it has been previously shown in other American groups. Some particular haplotypes in this Mataco tribe are described in this work.


Generic HLA-DRB1 gene oligotyping by a nonradioactive reverse dot-blot methodology. HLA-DRB1 allelic specificities can be determined using SSOs annealing to their complementary PCR-amplified target DNA. To perform HLA-DR oligotyping routinely for donors and recipients of bone marrow transplantation, a "reverse" dot-blot technique has been developed that consists in the hybridization of labeled PCR-amplified target DNA to SSOs that have been first attached to nitrocellulose membranes. The 15 oligonucleotides chosen enabled the following HLA-DRB1 "generic" specificities to be defined: DR1, BON, 2, 3, 4, 11, 11 JVM, 12, 13, 13 HAG, 14, 7, 8, 9, 10. The genomic DNA was amplified by asymetric PCR with incorporation of biotinylated deoxynucleotides predominantly to generate labeled single-stranded DNA. Hybridization between specific immobilized oligoprobes and target DNA was nonradioactively detected by a colorimetric reaction using alkaline phosphatase. The reverse dot-blot methodology was successfully tested, first, for the determination of HLA-DR4 subspecificities, and then the procedure was routinely applied to the generic HLA-DR oligotyping of bone-marrow donors and recipients.


Cytotoxic and proliferative allospecific T-cell clones contain perforin and mediate anti-CD3-induced cytotoxicity. Some in vitro-generated allospecific T-cell clones can kill target cells bearing specific antigen, whereas others can only proliferate in response to that antigen. The mechanism of target lysis by clones that exhibit antigen-specific cytotoxicity is thought to involve the exocytosis of lytic granules, which contain the pore-forming protein perforin. Here, CD4+, CD8+, and CD4-8- T-cell clones, positive for CD3 and the alpha/beta T-cell receptor, were tested for their ability to lyse the mouse-anti-human CD3 hybridoma OKT3; this hybridoma has been shown to trigger the cytolytic mechanism in cytotoxic T cells regardless of their clonal specificity. We found that all in vitro-generated allospecific T-cell clones can efficiently lyse the OKT3 targets whether or not they can kill alloantigen-bearing lymphoblastoid B-cell line targets. Furthermore, all tested clones contained perforin. The OKT3 hybridoma was not lysed by perforin-negative, CD3+ leukemic T-cell lines or by CD3- NK clones. Thus, the presence of perforin in T-cell clones correlated with their ability to lyse OKT3 targets, but not with their ability to lyse alloantigen-bearing targets. These results demonstrate that T-cell clones that are nonlytic when activated by specific antigen nevertheless contain a complete lytic mechanism and also support the proposed central role in perforin in that mechanism.


Congenital 21-hydroxylase deficiency as a new deletion mutation. Detection in a proband during subsequent prenatal diagnosis by HLA typing and DNA analysis. A child with 21-OH-def whose 9 weeks' pregnant mother was referred for prenatal diagnosis was found upon very careful histocompatibility testing to lack expression of any of his father's HLA antigens on his peripheral blood lymphocytes. The possibility of alternative paternity was considered to be extremely unlikely after additional genetic marker tests. The conclusion that the affected child's disease resulted from inheritance of a maternal CYP21B (21-OH) deletion and a de novo deletion in the paternal chromosome 6 segment that includes both the CYP21B (21-OH) and HLA genes was confirmed by subsequent DNA analysis using 21-OH, C4, DPB, and PCH6 probes. The presence of a heterozygous RFLP for DPB, the absence of a deletion for either CYP21B (21-OH) or C4 genes, and the presence of a paternal HLA antigen haplotype on the fetal cells additionally indicated that the fetus lacked the same deletion and could be predicted to be completely normal.


Conservation of genes encoding HLA-B5 and B35 cross-reactive group antigens in various races. HLA-B5 and B35 CREG antigens include HLA-B35, B51, B52, B53, and B78. Recent studies suggest that the genes encoding the HLA-B5, B35 CREG, and HLA-B58 antigens share a common ancestor. We sequenced the exons of the genes encoding HLA-B51, B53, and B58 from American black individuals and the gene HLA-B52 encoding from an Arabic individual, and compared them with previously reported sequences of HLA-B51 (B*5101) and HLA-B52 (B*5201) from Japanese, HLA-B53 (B*5301) from an Algerian, and HLA-B58 (B*5801) from a Sardinian. The sequences of the genes from the American black and Arabic individuals were identical to those from the other races. These findings support the hypothesis that these antigens have evolved prior to divergence of the major ethnic groups.


High yields of anti-HLA human monoclonal antibodies can be provided by SCID mice. In an attempt to develop a suitable model for increasing the yield of human anti-HLA mAbs, we have used mice with SCID for i.p. injection of two human-mouse heterohybridomas. HMP1 hybridoma secretes a DQB1*0201 allele-specific human mAb whereas HMP12 secretes a human mAb recognizing the DRB1*1101, 1102, 1103, and 1104 alleles. Both hybridomas could be grown in SCID mice as localized tumors with no apparent alteration in the morphology of the cells or in the immunoglobulin secretion. Ascitic fluid was produced that showed a 600- to 1000-fold increase in monoclonal antibody cytotoxic titer as compared with that obtained in tissue culture. HLA-DQB1* and DRB1* alleles recognized by ascites and supernatants from SCID-derived cultures were analyzed by microlymphocytotoxicity assay on a small panel of B-lymphoblastoid cell lines. The results show that HLA specificity was retained after in vivo passage.


Transfusion practices and costs in dogs. A geographically stratified sample of 25 small-animal practices administering at least six transfusions to dogs over the last 12 months was surveyed to determine how veterinarians obtain blood for transfusions, the direct costs of administering transfusions, and the impact of available blood on the management of critically ill dogs. The primary source of donor blood for each practice was a borrowed dog (12 practices) or in-house dogs kept on the premises (12 practices). Only one practice obtained blood from a nearby veterinary school. There was a wide variation in practices regarding testing for diseases and screening of donors. Thirty-six percent of practices surveyed did not screen dogs for infectious diseases or evaluate hematologic variables prior to blood donation. Twenty-four percent of the respondents evaluated the donors solely for the purposes of detecting microfilaria. The remaining 40% of the practices performed one or more of the tests generally recommended as part of a screening program for potential blood donors. The blood type of donors was determined in eight of the practices, whereas blood typing of recipients was not routinely performed. Ten of 25 practices performed blood crossmatches, but only one practice performed crossmatches in all cases. The distribution of direct costs per whole blood transfusion (500-ml unit) ranged from 25 to more than $300, with three fourths of the practices having costs less than $100. The higher-cost practices were those that maintained donors on the premises specifically for blood donation purposes.(ABSTRACT TRUNCATED AT 250 WORDS)


Investigation of an outbreak of velogenic viscerotropic Newcastle disease in pet birds in Michigan, Indiana, Illinois, and Texas. An outbreak of velogenic viscerotropic Newcastle disease in exotic pet birds in the United States from April through July 1991 was investigated. More than 2,000 pet birds in 5 states (Michigan, Indiana, Illinois, Texas, and Maryland) were tested. Infected birds were found in Michigan, Illinois, Indiana, and Texas. This disease was eradicated without spread to domestic poultry. Epidemiologic investigations revealed the source of infection to be double yellow-headed Amazon parrots suspected to be illegally imported into Texas.


Serum thyroid hormone concentrations and thyrotropin responsiveness in dogs with generalized dermatologic disease. Fifty-eight dogs with generalized dermatologic disease that had not been given glucocorticoids systemically or topically within 6 weeks of entering the study were evaluated for thyroid function by use of the thyrotropin-response test. Dogs were classified as euthyroid or hypothyroid on the basis of test results and response to thyroid hormone replacement therapy. Baseline serum thyroxine (T4), free T4 (fT4), and triiodothyronine (T3) concentrations were evaluated in the 58 dogs. Serum T4, fT4, and T3 concentrations were evaluated in 200 healthy dogs to establish normal values. Hormone concentrations were considered low if they were less than the mean -2 SD of the values for control dogs. Specificity of T4 and fT4 concentrations was 100% in predicting hypothyroidism; none of the euthyroid dogs with generalized skin disease had baseline serum T4 or fT4 concentration in the low range. Sensitivity was better for fT4 (89%) than for T4 (44%) concentration. Significant difference was not observed in serum T4 and fT4 concentrations between euthyroid dogs with generalized skin disease and healthy control dogs without skin disease. Serum T3 concentration was not accurate in predicting thyroid function; most of the euthyroid and hypothyroid dogs with skin disease had serum T3 concentration within the normal range.


Comparison of computed tomography and radiography for detecting changes induced by malignant nasal neoplasia in dogs. The ability of computed tomography and radiography to detect changes associated with nasal neoplasia was compared in dogs. Eighteen areas or anatomic structures were evaluated in 21 dogs for changes indicative of neoplasia. Computed tomography was superior (P < or = 0.05) to radiography for detecting changes in 14 of 18 areas. Radiography was not superior for detecting changes in any structure or area. Computed tomography reveals vital information not always detected radiographically to assist in providing a prognosis and in planning treatment for nasal neoplasms in dogs.


Characteristics of pet populations and households in the Purdue Comparative Oncology Program catchment area, 1988. A 1-stage, random-digit dial telephone survey was conducted to obtain information on characteristics of pet populations and pet-owning households in 1988 in Marion and Tippecanoe Counties, Indiana. Interviews for 653 out of 731 eligible households were completed (response rate, 88%). Approximately half of the households in each county owned a pet. Of these, 35% owned at least 1 dog, and 23% owned at least 1 cat. Households with pets were more likely to be larger and have a higher median income score than were households without pets. Households with children between 6 and 17 years old were more likely to own pets than were households with no children; however, no difference in pet ownership proportions was determined for households with children < or = 5 years old, compared with households without children. For dogs, younger dogs and male dogs were less likely to have been neutered than older dogs and female dogs. Older cats were more likely to have been neutered than younger cats, with neutering percentages of > 90% for cats in the oldest age group. Approximately 20% of dogs and 40% of cats had not been seen by a veterinarian in the 12 months preceding the interview.


Serum haptoglobin concentration in swine naturally or experimentally infected with Actinobacillus pleuropneumoniae. Serum haptoglobin (Hp) concentrations were measured in swine that were naturally or experimentally infected with Actinobacillus pleuropneumoniae. In swine from a specific-pathogen-free herd, mean serum concentration of Hp (+/- SD) was 5.79 +/- 1.06 mg of cyanmethemoglobin-binding capacity (CHBC)/dl. Serum Hp concentrations in paired samples were measured at 7-day intervals in 40 swine randomly selected from a conventional herd that was experiencing an acute episode of pneumonia and deaths caused by A pleuropneumoniae serotype-5 infection. Day-0 and -7 serum Hp concentrations were 24.58 +/- 1.38 and 23.10 +/- 1.12 mg of CHBC/dl, respectively, with no significant difference between these measurements. In a second conventional herd with a history of chronic infection with A pleuropneumoniae serotype 5, serum concentrations of Hp measured in paired samples obtained 6 days apart were 12.36 +/- 0.81 and 18.63 +/- 0.76 mg of CHBC/dl, respectively, and were significantly (P < 0.05) different from each other. Twenty-nine 12-week-old conventional swine were challenged intranasally with A pleuropneumoniae serotype 1 (n = 19) and serotype 5 (n = 10). Serum Hp concentration increased from prechallenge concentrations of 7.49 +/- 1.38 and 15.10 +/- 1.22 mg of CHBC/dl, respectively, to 41.01 +/- 1.35 and 22.37 +/- 1.78 mg of CHBC/dl, respectively, 72 hours after challenge. For these 29 swine, serum Hp concentration was positively correlated with rectal temperature (r = 0.34; P < 0.001) during the immediate postchallenge period.


Gastric perforation associated with administration of ibuprofen in a dog. A 3-year-old Chow Chow was examined because of a 2-week history of vomiting and anorexia after administration of 200 mg of ibuprofen. Peritoneal effusion and free gas within the peritoneal cavity were observed on radiography of the abdomen. A full-thickness perforation of the pyloric antrum and pylorus were detected during exploratory laparotomy, and a Billroth-I gastroduodenostomy was performed successfully.


Chronic diarrhea associated with intestinal anomalies in a six-year-old dog. A 6-year-old mixed-breed dog was referred because of chronic diarrhea, which had begun after antibiotic treatment for pulmonary disease. Intestinal bacterial overgrowth was presumptively diagnosed on the basis of intestinal mucosal cytologic examination and low serum cobalamin concentration. When the diarrhea did not respond to treatment, contrast radiography was performed and a short small intestine was found. The diarrhea could not be managed by medical treatment, and the dog was euthanatized. Necropsy revealed 2 blind intestinal pouches attached to a 46-cm length of small intestine connecting the stomach and colon. The dog's deteriorating condition was attributable to necrosis and rupture of 1 of the pouches.


Thrombocytopenia associated with administration of trimethoprim/sulfadiazine in a dog. A diagnosis of trimethoprim/sulfadiazine-induced, immune-mediated thrombocytopenia in a dog was made, using a novel in vitro assay for thrombolytic activity. The assay quantifies thrombolytic activity by measuring the amount of platelet fragments in normal canine platelets before and after incubation with plasma from the thrombocytopenic dog. This report confirms previous reports of the development of thrombocytopenia after administration of trimethoprim/sulfadiazine, and describes a new assay that, after further validation, may be useful in the diagnosis of immune-mediated thrombocytopenia when an adequate sample of platelets cannot be obtained for quantification of platelet-associated IgG.


Obstructive enterolith in an 11-month-old miniature horse. An obstructive enterolith was diagnosed in an 11-month-old 68-kg miniature horse with a 24-hour history of mild, intermittent colic. Abdominal radiography revealed gas distention of the cecum and the right and left ascending colon, and a radiodense elliptical structure in the area of the left kidney. During exploratory laparotomy, the right dorsal colon was noticed to contain ecchymotic hemorrhage and generalized discoloration in the portion proximal to the location of the enterolith. While exteriorizing the ascending colon, it ruptured caudal to the diaphragmatic flexure in the right dorsal colon. Radiography of the enterolith did not reveal a metallic center, but division of the stone allowed identification of a canvas cloth, and chemical analysis determined the stone's composition to be ammonium magnesium phosphate. Although equine enteroliths were reported over 100 years ago and the factors involved in their formation were investigated, the reasons for their development have yet to be determined. The mean reported age of horses requiring abdominal surgery because of an obstructive enterolith is 10 years, with the youngest being 4 years. The time required for an enterolith to enlarge is unknown, although a true enterolith has not been reported in horses younger than 4 years of age.


Calcifying tendinopathy of the biceps brachii in a dog. Calcifying tendinopathy of the biceps brachii was associated with lameness in a 1.5-year-old Rottweiler. Lameness was relieved by excision of the calcified mass and suture repair of the partially ruptured tendon. Calcifying tendinopathy is not well described in dogs, but it is a recognized clinical syndrome in human beings. The etiopathogenesis of the condition in human beings and dogs is poorly understood. Wider recognition of calcifying tendinopathy in dogs should lead to a better understanding of the disease and development of appropriate clinical treatments.


Results of surgical management of portosystemic shunts in dogs: 20 cases (1985-1990). Serum bile acid (SBA) values were obtained in 20 dogs, 2.5 to 60 months (mean, 18.6 months) after surgery for occlusion of a congenital portosystemic shunt. The SBA values obtained after food was withheld were significantly (P < 0.005) reduced at follow-up, compared with preoperative values. In 85% of the dogs (17/20), response to surgery was graded by the owners as excellent or good, although most dogs had abnormal SBA values at follow-up. Significant difference in clinical results or follow-up SBA values was not found in dogs that had complete or partial occlusion of the shunting vessel. Correlation between clinical results and SBA values also was not detected. Dogs that were > 2 years old at the time of diagnosis and surgery had a significantly (P = 0.012) poorer clinical result than that of dogs < 1 year old.


Evaluation of risk factors for blastomycosis in dogs: 857 cases (1980-1990). An epidemiologic study was conducted by use of the Veterinary Medical Data Base to investigate risk factors for blastomycosis in dogs. From January 1980 through June 1990, 971 cases of blastomycosis in dogs from 22 North American veterinary teaching hospitals were identified. Of these cases, 114 (11.7%) were excluded from the study because of incomplete information regarding age, body weight, sex, and neuter status. A control group of 417,079 dogs was selected that included all other dogs with medical conditions unrelated to blastomycosis for which records were submitted to the data base during the same period. The prevalence of blastomycosis in dogs was 205/100,000 admissions during the study period. When veterinary teaching hospitals were grouped on the basis of their general geographic location, dogs in the East South central, East North central, West South central, and South Atlantic regions had a significantly (P < 0.05) increased risk of acquiring blastomycosis, compared with that of dogs in the Mountain/Pacific region. When teaching hospitals from all geographic regions were considered, dogs had a significantly (P < 0.05) increased risk of acquiring blastomycosis in autumn, compared with that in spring. Sporting dogs and hounds, as defined by the American Kennel Club, were at increased risk for blastomycosis. At highest risk were Bluetick Coonhounds, Treeing-walker Coonhounds, Pointers, and Weimaraners, compared with mixed-breed dogs. Ages of dogs with blastomycosis tended to be normally distributed. Generally, the highest-risk group was composed of sexually intact male dogs, 2 to 4 years old, weighing 22.7 to 34.1 kg. This same pattern was observed for sporting dogs and hounds.(ABSTRACT TRUNCATED AT 250 WORDS)


Traumatic luxation of the cubital joint (elbow) in dogs: 44 cases (1978-1988). The medical records from 44 dogs with traumatic luxation of the cubital joint were reviewed. Closed reduction was performed in 35 dogs, and open reduction in 9 dogs. Excellent or good results were achieved in 31 of 35 dogs (88.6%) treated by closed reduction and in 5 of 9 dogs (55.6%) treated by open reduction. Collateral ligament repair, performed in 5 dogs, did not appear to affect the prognosis. Factors such as body weight and type and duration of external coaptation also did not appear to influence the outcome. Increased dysfunction associated with open reduction most likely was caused by the chronic nature of the dislocation, iatrogenic damage to the articular cartilage during reduction, or both. A good prognosis for return to normal function with minimal gait abnormality can be expected in most cases of traumatic luxation of the cubital joint treated by early closed reduction.


Flavobacterium indologenes infection in leopard frogs. An investigation of an epidemic of infectious disease in a frog (Rana pipiens) colony was conducted. Six of 40 frogs in a continuous (once through) water flow housing system had weight loss, swollen abdomen, corneal edema, uveitis, subcutaneous edema, petechial hemorrhage, incoordination, and respiratory distress. The frogs had lesions consistent with bacterial septicemia. A gram-negative, nonfermenting bacillus, Flavobacterium indologenes (Flavobacterium sp biovar IIb), was isolated in pure culture from tissues and blood. The clinical isolate was used to inoculate healthy frogs sc. An isolate identical to the one isolated from the sick frogs was recovered from tissues and blood of the inoculated frogs. Inoculation of the housing water in a nonflow-through system did not result in disease, despite proliferation of the Flavobacterium spp in the water; therefore, it is likely that establishment of infection requires the presence of the organism in sufficient numbers and a portal of entry into the body.


Perineal hernia in a cougar.

Perineal hernia in a cougar. An 8-week-old female cougar with a history of acute perineal swelling was determined to have a perineal hernia. The hernia was repaired with the conventional suture technique. This case represents an unusual perineal hernia, which may have been congenital.


Androgen regulation of programmed death of normal and malignant prostatic cells. Androgen-dependent normal prostatic glandular cells and androgen-dependent prostatic cancer cells can be induced to undergo cell death after androgen ablation. This death does not require the cells to proliferate and occurs as an energy-dependent process collectively referred to as "programmed cell death" in which the cells actively commit "suicide." Associated with this programmed cell death pathway is the enhanced expression of a series of genes and the fragmentation of the genomic DNA into nucleosomal oligomers. This genomic DNA fragmentation is the irreversible commitment step in the death of the cell and results from activation of Ca2+/Mg(2+)-dependent endonuclease activity within the cell nucleus. This activation is due to sustained elevation of intracellular free Ca2+ (Cai) induced after androgen ablation. Metastatic prostatic cancer within an individual patient is heterogeneous, including both androgen-dependent and -independent cancer cells. Thus, androgen ablation is rarely curative since it only induces the programmed death of the androgen-dependent cancer cells without activating this pathway in the androgen-independent cancer cells within the patient. Androgen-independent prostatic cancer cells do not activate this death process after androgen ablation, since this does not induce a sustained increase in Cai. A new approach to treat androgen-independent prostatic cancer cells has focused on the use of chemotherapeutic agents to induce a sustained increase in Cai. These studies demonstrate that if such a sustained elevation in Cai is maintained, even androgen-independent prostatic cancer cells undergo programmed cell death.


Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors. Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.


Role of nerve growth factor-like protein in the paracrine regulation of prostate growth. Prostate cancer is the most commonly diagnosed form of malignant neoplasia in men. Considerable evidence has accumulated suggesting that paracrine interactions between stromal cells and epithelial cells mediate, in part, the growth and development of the prostate. A nerve growth factor-like protein secreted by stromal cells has been implicated in the paracrine regulation of prostate epithelial tumor cell growth in vitro. This prostate-derived nerve growth factor-like protein differs from the known members of the neurotrophin family of proteins, and may represent a prostate-specific form of this family of gene products. Furthermore, corresponding nerve growth factor receptors have been localized to the epithelial cells of the human prostate in vivo, consistent with a role of the receptors and the adjacent nerve growth factor-like protein secreted by stromal cells, in the paracrine regulation of prostate growth and neoplasia.


Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive system in the rat. The effects of carbendazim (methyl 2-benzimidazole carbamate) on the testis, efferent ductules, and sperm were determined in the adult rat after a single oral dose. Two experimental trials were performed: a time response between 2 hours and 32 days after exposure using 0 and 400 mg/kg, and a dose response at 2 and 70 days after exposure using 0 to 800 mg/kg doses. In experiment 1, effects were seen throughout the 32-day period, beginning 8 hours after exposure; the effects included first an increase in testis weight, then decreases in testicular spermatid numbers and in the percentage of morphologically normal cauda sperm. In experiment 2, significant testicular and efferent ductal alterations occurred in animals treated with doses of 100 mg/kg or greater. A dose-dependent increase in testicular weight 2 days after treatment was accompanied by increases in seminiferous tubular diameter and excessive loss of immature germ cells in a stage-dependent manner. There was also a dose-dependent increased incidence of occlusions in the efferent ductules. The occluded ductules were characterized by severe inflammation and exhibited disorganization of the epithelium. At 70 days, there were dose-dependent decreases in mean testis weight and mean seminiferous tubular diameter; however, only minimal long-term effects were seen at 50 mg/kg. In testes exhibiting seminiferous tubular atrophy of greater than 25% (100 mg/kg or greater doses), all of the testes were associated with efferent ductules containing occlusions. Caput sperm numbers were significantly reduced in these testes. Occlusions, abnormal ductules, fibrosis, spermatic granulomas, and mineralization were observed in the ductuli efferents. Long-term effects of carbendazim on the testis were induced primarily by ductal occlusions. Results show that carbendazim produces more severe short- and long-term effects on the male reproductive system than the fungicide benomyl.


Effect of fucoidin on human sperm-zona pellucida interactions. The authors recently reported that fucoidin (a polymer of predominantly L-fucose sulfate) produced a strong, significant, and dose-dependent inhibition of sperm-zona binding under hemizona assay conditions. The current studies were designed to evaluate the mechanisms underlying this inhibitory activity. Using computerized semen analysis, the monoclonal anti-sperm antibody T-6 and indirect immunofluorescence technique, and the fura-2 indicator, no significant impact of fucoidin on sperm motion parameters, on the spontaneous acrosome reaction, or its prerequisite, the increase in calcium influx, were observed. Subsequently, a mild acid hydrolysis of the fucoidin molecule was performed, followed by sizing hydrolysates in a Biogel P2 column and separating fractions (n = 6). All fucoidin fragments significantly inhibited tight binding of human sperm to human zona pellucida under hemizona assay conditions (range, 56%-94%) when tested individually. These results provide further evidence that the effect of fucoidin is produced by a receptor-ligand association.


Differential regulation in the release of bioactive versus immunoactive gonadotropins from cultured rat pituitary cells by inhibin and androgens. Using in vitro bioassays and radioimmunoassays, the authors examined the interactions between purified 32kD ovine inhibin, testosterone, and dihydrotestosterone on basal and GnRH-stimulated secretion of gonadotropins in primary rat pituitary cell cultures. This study demonstrates that inhibin and androgens: 1) differentially regulate gonadotropin levels, 2) cause changes in the amounts of bioactive and immunoactive FSH and LH released. In conclusion, differences in bio- and immuno-FSH and LH secretion suggest that the endocrine milieu results in not only quantitative but qualitative changes in the secreted gonadotropin isoforms.


Androgen-induced prevention of the outgrowth of cranial gonadal suspensory ligaments in fetal rats. Normal and disturbed testicular descent is frequently approached exclusively through a consideration of the caudal testicular suspensory apparatus. This is surprising, because embryonal gonads develop with both cranial and caudal suspensory ligaments, and the sexes differ with respect to the persistence and development of both the cranial and the caudal ligaments. The current study examined the possible role of fetal testicular androgens in male-specific failure of the development of the cranial gonadal suspensory apparatus in rats. Normal male fetuses were studied, as well as fetuses exposed to the anti-androgen flutamide from day 10 after conception. Females were given daily injections of methyl-testosterone alone or in combination with the anti-androgen cyproterone acetate from day 15 after conception, and were studied on day 22. The cranial ligaments remained of minor extension in normal males but developed considerably in females. They developed in female fashion in males exposed to flutamide, and persisted throughout postnatal life. Cranial ligaments did not develop in females that had been exposed to methyl-testosterone. Simultaneous treatment with a large dose of cyproterone acetate effectively counteracted this effect. Fetal testicular testosterone thus appears to play a key role in the prevention of the outgrowth of the cranial gonadal/genital ligament in rats. The supposed function of this suspensory apparatus makes it likely that its persistence in males, as the consequence of inappropriate androgen action during fetal life, facilitates disturbance of testicular descent. This finding may contribute to understanding developmental disorders underlying disturbed testis descent in humans.


Dose-response effects of gonadotropin-releasing hormone on plasma concentrations of gonadotropins and testosterone in fertile and subfertile stallions. Five fertile and five subfertile stallions were treated with a single intravenous injection of saline the first week followed by a single intravenous injection of varying doses of gonadotropin-releasing hormone (5, 10, 25, 100, 500 micrograms) given in a randomized fashion over the next 5 weeks during the nonbreeding season. Blood samples were collected periodically before and after treatment for analysis of luteinizing hormone, follicle stimulating hormone, and testosterone content by radioimmunoassay. Before treatment, semen samples were collected every other day for 3 weeks for analysis of volume, concentration, motility, pH, and morphology. Basal plasma levels of luteinizing hormone were higher (P < 0.05) in the subfertile group, follicle stimulating hormone levels tended to be higher (P < 0.10) in the subfertile group, and testosterone levels were similar in the two groups. A significant linear-log dose-response relationship was observed for plasma luteinizing hormone (P < 0.05) and follicle stimulating hormone (P < 0.05) to exogenous gonadotropin-releasing hormone in both the fertile and subfertile group. A linear-log dose-response relationship was also observed for plasma testosterone (P < 0.05) in the fertile group. The magnitude of the luteinizing hormone and follicle stimulating hormone response to gonadotropin-releasing hormone across doses was similar in both groups of stallions. A significant testosterone response to gonadotropin-releasing hormone in the subfertile group of stallions was not observed (P > 0.05). Mean testosterone concentrations after treatment in terms of net increase and percent of baseline were significantly lower (P < 0.05) in the subfertile group compared to the fertile group.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructure of adult and juvenile marmoset (Callithrix jacchus) Sertoli cells in vivo and in vitro. The morphologic differentiation of Sertoli cells isolated from adult and juvenile marmosets and cultured on different extracellular matrices was evaluated by light and electron microscopy and compared to cells in vivo. Both cell types could be maintained in culture for at least 6 days. The degree of cellular differentiation, shape, ultrastructural appearance, and polarity seemed to benefit from laminin-coated substrata, compared with collagen-, fibronectin-, serum-, and heparan sulfate-coated substrata. With the former two substrates, a difference in behavior between juvenile and adult cells was evident. Whereas juvenile cells displayed a lesser degree of differentiation, adult cells exhibited identical morphologic characteristics in culture and in vivo. Cyclicity of morphologic features was not found, neither in vivo nor in vitro. The results indicate that: (1) laminin plays a unique role for marmoset Sertoli cell differentiation in vitro compared with other extracellular components; (2) a greater similarity between cells in vivo and in vitro is evident with adult Sertoli cells; and (3) the adult marmoset monkey could provide a primate model for mature Sertoli cells in culture, since there is a close similarity to human adult Sertoli cells in vitro and in vivo.


Human sperm acrosin. Further studies with the clinical assay and activity in a group of presumably fertile men. The goals of this study were to determine the effect of the nonionic detergent, Triton X-100, on the recovery of acrosin in the clinical assay (Kennedy et al, 1989), since previous investigations have used higher concentrations for acrosin extraction from spermatozoa; and to establish the minimal acrosin activity in fertile men. The recovered acrosin activity was dependent on the concentration of Triton. A peak in acrosin activity was obtained at 0.01% to 0.02% Triton, the approximate critical micelle concentration (CMC; 0.015%). The bimodal effect of Triton was not due to substrate/buffer alterations, to the degree of acrosomal disruption as assessed by light and transmission electron microscopic examination, or to its effect on the kinetic properties of acrosin, as determined by spectrophotometric analysis of acid-extracted enzyme. However, Triton affected the conversion of proacrosin to acrosin, with peak activation occurring at 0.01% to 0.02% detergent. The acrosin activity of a group of presumably fertile men (as established by the production of offspring under natural conditions) varied from 18 to 42 microIU/10(6) spermatozoa, as assessed by the clinical assay containing 0.01% Triton. Furthermore, men who had initial acrosin values in the low normal range (18 to 25 microIU/10(6) sperm) were observed for 11 months. The acrosin activity of their ejaculates never fell below 17 microIU/10(6) sperm. Thus, it can be tentatively assumed that the minimal levels of acrosin for naturally fertile men are 17 to 18 microIU acrosin/10(6) sperm in this assay.


The detection of alcohol problems in a primary care clinic. The CAGE is a four item questionnaire which is used to help clinicians identify alcohol problems. Charts of 433 primary care patients who were given a medical health form containing the CAGE questions (experimental patients) were compared with charts of 451 patients given a similar form that did not contain the CAGE questions (control patients). Alcohol problems were detected more frequently in the experimental patients (10.6%) than in the control patients (6.7%) (p < 0.05). This difference in detection tended to be most evident for persons with milder alcohol problems (problem drinking) as opposed to more well developed alcohol abuse. Experimental patients (3.7%) also tended to be more likely than control patients (2.9%) to receive active alcohol treatment during their initial medical visit. Medical health screening forms which include the CAGE questions may promote the identification of alcohol problems in primary care.


Legislative interventions to increase access to screening mammography. Although numerous studies have established that breast cancer mortality can be significantly reduced through early detection, only a small percentage of women obtain screening mammograms at intervals recommended by the National Cancer Institute, the American Cancer Society, and other major medical organizations. This paper examines the importance of cost as a barrier to routine screening and the state legislative movement to make screening mammography a basic health insurance benefit. Mammography "knowledge, attitudes, and behavior" studies offer conflicting findings on the extent to which cost enters into the decision to have a mammogram. Women seldom report cost as a major reason for postponing or failing to have a mammogram; yet, descriptive studies show a consistently positive relationship between income and mammography use. State mammography reimbursement laws vary greatly with respect to whether screening mammography is a required or optional benefit, payment limits, and eligibility and referral requirements. Although state-specific data on the percentage of women with private health insurance are not available, 1987 National Medical Expenditure Survey estimates for U.S. Census geographic divisions suggest that the New England, East North Central, West North Central, Middle Atlantic, and Mountain states have the highest percentages of women who are privately-insured and, thus, potentially eligible for legislated mammography benefits. Access to screening mammography also is likely to be influenced by the proportion of employer-sponsored health plans that are self-insured and, therefore, exempt from minimum benefit mandates and the extent to which women are aware of the screening coverage.


Prevalence of smoke detectors and safe tap-water temperatures among welfare recipients in Memphis, Tennessee. The poor are at high risk for fire- and scalding-related injuries and deaths, many of which could be prevented by installing smoke detectors and reducing residential tap-water temperatures to safe levels. The goal of this study was to measure the prevalence of smoke detectors and safe tap-water temperatures among welfare recipients and compare subjects living in safe and unsafe homes. We interviewed 109 black women residing in inner-city Memphis who were receiving Aid to Families with Dependent Children and inspected their homes for functional smoke detectors and safe tap-water temperatures. Of the 533 residents reported to live in the homes visited for this study, 53.1% were not protected by smoke detectors. More crowded residences were substantially more likely to lack detectors. Most of the smoke detectors which were present were provided by landlords. Of 69 smoke detectors examined, 17.4% did not work. Of the 153 children age 0-5 years living in the homes we visited, 79.1% lived in residences with excessively hot tap water (> 54 degrees C). We conclude that additional efforts are needed to increase the use of smoke detectors and to decrease excessively hot tap water in the homes of welfare recipients.


Modulation of immunoradiometric and bioactive follicle stimulating hormone secretion and clearance in young and elderly men during treatment with tamoxifen or flutamide. The secretion and clearance of immunoactive and bioactive follicle-stimulating hormone (FSH) in healthy young men (N = 10) and elderly men (N = 7) during blockade of endogenous sex steroid hormones with tamoxifen, an antiestrogen, and flutamide, an antiandrogen, was investigated. To this end, subjects underwent blood sampling basally every 10 minutes for 24 hours, and then received 2 consecutive intravenous pulses of synthetic gonadotropin releasing hormone (GnRH; 10 micrograms and 100 micrograms) every 2 hours. This paradigm was repeated on two subsequent visits, in which subjects received either flutamide HCl, a specific nonsteroidal competitive antagonist of the androgen receptor (750 mg daily for 3 days), or tamoxifen, a selective antagonist of the estrogen receptor (20 mg daily for 9 days). Serum immunoactive FSH concentrations were measured in each sample by immunoradiometric assay (IRMA). Serum bioactive FSH concentrations were determined by an in vitro bioassay (rat granulosa cell aromatase system) on 24-hour serum pools. Deconvolution analysis was used to analyze both the FSH IRMA 24-hour time series and FSH release after GnRH. Comparisons between young and elderly men of the basal state showed significantly increased 24-hour mean serum immunoactive and bioactive FSH concentrations and significantly decreased free testosterone concentrations in elderly men. By deconvolution analysis, elderly men had a significant decrease in FSH secretory burst duration, and an increase in FSH half-life and FSH secretory burst amplitude compared with younger men. In response to sex steroid receptor blockade in young men, there was a significant increase in mean serum bioactive FSH concentrations during antiandrogen treatment, but not during antiestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Student nurse opinions about the importance of health promotion practices. Senior nursing students (N = 505) attending 13 schools in the NY metropolitan area were administered a questionnaire to assess their beliefs about the importance of health promotion behaviors to the average person. Respondents rated 23 health promotion practices on a Likert scale. The five most important items concerned: knowledge of drug contents and their side effects; the elimination of cigarette and cigar smoking; eating a balanced diet; and using a seat belt. The least important item concerned having an annual exercise test. These results were similar to those found for other health care providers, including physicians, dietitians, and pharmacists. Further, the results were similar to those found in an earlier survey of student nurses. Nurses' attitudes and beliefs seem supportive of health promotion and disease prevention. These attitudes also seem associated with the nurse's role as health promoter. Positive nurse attitudes/beliefs toward health promotion are posited as precursors to the alteration of patient behaviors, although further exploration in this area is needed.


Food commercials during television soap operas: what is the nutrition message? Studies have examined food commercials appearing during daytime, prime time, and Saturday morning programming; none have evaluated commercials aired during soap operas. The purposes of this study were to examine the dietary composition of food products advertised during soap operas and the health and nutrition claims made on their behalf, using current dietary recommendations as a basis of evaluation. A total of 508 commercials videotaped during the top nine daytime serials for five consecutive days, were for food or beverage products. Most foods advertised were rated low in sugar, fat, sodium, and dietary fiber, and health and/or nutrition appeals occurred frequently. However, of the foods advertised as "low cholesterol," 77% were high in fat. Similarly, those foods advertised as low in saturated fat were high in total fat content. Forty-three percent of the food commercials promoting nutrition were for items such as flavored drinks with little nutrient value. Generally, the nutrition messages conveyed in these commercials supported current dietary recommendations, but the way in which the food products were promoted was often inconsistent and confusing, particularly in the area of fat. Nutrition educators need to address these inconsistencies when assisting consumers in interpreting television food advertising messages.


Structure, function and possible clinical application of transforming growth factor-beta. Transforming growth factor-beta (TGF-beta) is a family of multifunctional 25 kDa proteins. TGF-beta was originally identified because of its ability to induce the growth of normal rodent fibroblasts in soft agar, but is now known as a potent growth inhibitor for many different cell types. In addition, TGF-beta is known to regulate the differentiation of cells, induce chemotaxis of cells, and to induce the accumulation of extracellular matrix proteins. In vivo, TGF-beta stimulates the repair of soft as well as hard tissues. It also acts as a potent immunosuppressant. TGF-beta is produced as latent high molecular weight complexes; since it is produced by many different cell types, and most cells have receptors for TGF-beta, the activation of latent TGF-beta is likely to be an important step in the regulation of its action. TGF-beta exerts its effects by binding to specific cell surface receptors. The type I and type II TGF-beta receptors are suggested to be the most important for signal transduction; a recent report has disclosed that the type II receptor has a serine/threonine kinase domain. Since TGF-beta is a potent growth regulator with multifunctional activity, it may be useful in the treatment of certain clinical disorders. Local application of TGF-beta is shown to accelerate wound healing. Since an increase in TGF-beta activity is often observed in various fibrotic disorders, antagonists for TGF-beta might be valuable in the treatment of such diseases.


The growth regulation of keratinocytes.

The growth regulation of keratinocytes. Taken together, all these results suggest that TGF-alpha and amphiregulin are the major growth factors for positive growth control, although we don't know which plays the central role. For negative growth control, TGF-beta is the major factor. However, it remains to be solved which isoform of TGF-beta is naturally involved in keratinocyte growth regulation.


Stimulation of keratinocyte migration by growth factors. Migration of keratinocytes from the wound edge is thought to be one of the critical features of reepithelialization. A quantitative migration assay was carried out using normal human keratinocytes. Keratinocytes, seeded on 12 well plates, were grown in serum free, keratinocyte growth medium (KGM, Curabo Co) with 0.08 mM Ca2+. The medium was switched from KGM to keratinocyte basal medium (KBM) 6 h prior to the wounding. Half of the plate's confluent monolayer of keratinocytes was removed with razor blade, and the remaining keratinocytes were incubated in KBM for 16 hrs in the presence of indicated growth factors. After incubation, the cells were fixed and counted at 100 magnification. Migration was quantitated by counting the number of cells in ten successive 125-microns zones. Transforming growth factor alpha (TGF-alpha), acidic and basic fibroblast growth factor (aFGF, bFGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and insulin-like growth factor-I (IGF-I) stimulated the migration of keratinocytes, while TGF-beta suppressed it.


The effects of basic fibroblast growth factor and doxorubicin on cultured human skin fibroblasts: relevance to wound healing. The effects of basic fibroblast growth factor (bFGF) and doxorubicin on cultured human skin fibroblasts were examined in order to determine their relevance to wound healing. bFGF is shown to stimulate fibroblast collagenase production per cell, and this effect in vitro seems to be one explanation for its efficacy in wound healing. Doxorubicin inhibited not only fibroblast proliferation but also collagen production by inactivating prolyl hydroxylase. This result may explain the reduced wound healing in patients undergoing treatment with doxorubicin. These studies indicate the importance of assessing the effects of growth factors on matrix metabolism in order to understand their roles in wound healing.


Growth factors and chronic wounds: the need to understand the microenvironment. The care of chronic wounds has become a major health issue in developed countries because of their increasingly elderly populations. There is hope that progress made in understanding and producing growth factors will lead to their successful use to induce faster and better healing of chronic wounds. This report will discuss growth factors in the context of their use in chronic wounds, and will focus on the importance of the wound microenvironment in determining the interactions between growth factors and wounds. We believe that a greater understanding of the chronic wound microenvironment will be of benefit in the optimal use of growth factors. In published studies, we have found that wound fluid taken from acute wounds stimulates fibroblast and endothelial cell proliferation, whereas fluid obtained from chronic non-healing wounds inhibits the growth of fibroblasts, endothelial cells, and keratinocytes. In this report, we describe the effect of these two types of wound fluid on the synthesis of extracellular matrix components. We hypothesize that the chronic wound microenvironment is generally non-conducive to cell growth, and that this may prevent a truly successful use of topical growth factors in chronic wounds. Novel approaches in the delivery of growth factors to wounds may be necessary to overcome these obstacles.


A wound healing model using healing-impaired diabetic mice. A quantitative histological approach was employed to evaluate the effects of basic fibroblast growth factor (bFGF) in healing-impaired diabetic mice. The dorsal areas of female mutant diabetic mice, C57BL KsJ db/db (Jackson Lab.), were given two 6 mm-size full thickness wounds with a punch biopsy instrument. After application of bFGF, the wounds were left open. 8 days after wounding, the mice were sacrificed, and histological sections were evaluated using several histological parameters, such as the degree of wound closure, granulation tissue thickness, matrix density, and capillary numbers. Application of 5 micrograms of bFGF for 5 days induced significant responses by all of these dermal parameters when compared to those of non-treated db/db mice (p < 0.001). A minimum of 0.5 micrograms bFGF per day was required for a significant effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 days and decreased after 12 days, while matrix density continued to increase until the 18th day.


The role of growth factor in wound healing. A pig wound healing model was developed to study the morphological processes involved in incisional and excisional wound healing, the immunohistochemical localization of TGF-alpha in wound healing, and the latter's relevance. In incisional wounds, a few layers of epidermis were regenerated after only 32 hours. In contrast, several layers of epidermis were regenerated on day 7 in excisional wounds. In general, the incisional wound model is useful for evaluating tensile strength, while the excisional wound model is suitable for evaluating epidermal regeneration. Immunohistochemical study showed that keratinocytes migrating from wound edges expressed TGF-alpha very faintly, while TGF-alpha was present prominently in the upper several layers of the epidermis adjacent to the wound, which resembled normal epidermis and showed no changes during the wound healing process.


Molecular bases for hereditary cancer-prone diseases. Constitutional loss or inactivation of one copy of a tumor-suppressor gene, as exemplified by hereditary retinoblastoma, increases the propensity for malignancies by reducing the number of events necessary for the complete loss of the negative regulatory function. We developed a selectable mutation assay employing a human lymphoblastoid cell line (LCL) derived from a heterozygous carrier of 2,8-dihydroxyadenine urolithiasis, adenine phosphoribosyltransferase (APRT) deficiency, for dissecting the second step in loss-of-function mutations and for determining the potential of physical and chemical agents for producing such mutations. The mode of mutational events arising in the wild-type allele of the functionally heterozygous APRT gene resembled that reported for tumor-suppressor genes in malignancies in that mitotic non-disjunctions or recombinations as well as deletions prevailed. Ultraviolet light (UV) was much less efficient in inducing these types of mutations than ionizing radiation. A group of autosomal recessive cancer-prone diseases, including xeroderma pigmentosum (XP), has been characterized as being more susceptible to genomic insults, owing to some defects in DNA processing, such as replication, repair, or recombination. This increased genomic instability may accelerate the gain-of-function mutation at a proto-oncogene and/or the loss-of-function mutation at a tumor-suppressor gene. XP complementation group A (XP-A) LCLs were extremely sensitive to UV-mutagenesis at the hypoxanthine phosphoribosyltransferase (HPRT) locus even at equicytotoxic doses. Some unique mechanism may operate in UV-mutagenesis in XP-A. We have succeeded for the first time in rendering XP-A cells tumorigenic in athymic mice by applying multiple exposures to UV and subsequent treatment with TPA.(ABSTRACT TRUNCATED AT 250 WORDS)


Xeroderma pigmentosum: recent clinical and photobiological aspects. In Japan, more than 400 patients with xeroderma pigmentosum (XP) have been registered. The major groups are XP-A and variant, while clinically mild types of XP with intermediate levels of unscheduled DNA synthesis (UDS) have recently been increasing. The classical type of XP-A and some of the XP-D patients exhibit neurologic abnormalities. XP individuals display a marked increase in the frequency of skin malignancy. Development of skin malignancies appears to be related to the level of DNA repair capacity; the lower the capacity, the earlier and more frequently the skin tumors develop. Furthermore, the incidence of internal malignancy in XP patients is at least ten times higher than that for the Japanese general population over the age of 40 years. Cultured fibroblasts from XP patients exhibit higher sensitivity not only to UVC but also to UVB. The cellular sensitivity to UVB may correlate to photosensitivity in vivo from a study on a group E patient who showed age-related changes in photosensitivity and cellular sensitivity to UVB. We have also reviewed current status of molecular genetics in XP.


Alterations of mitogenic responses of mononuclear cells by arsenic in arsenical skin cancers. We have studied the endemic occurrence of chronic arsenism in a limited area on the southwest coast of Taiwan. The effects of arsenic on the mitogenic responses of mononuclear cells (MNC) derived from patients with arsenical skin cancers in that area were evaluated. The subjects enrolled in this study included patients with 1) Bowen's disease, 2) arsenical skin cancers (basal cell carcinoma and squamous cell carcinoma), 3) non-arsenical skin cancers (basal cell carcinoma and squamous cell carcinoma), 4) nasopharyngeal cancer and 5) healthy controls from endemic and non-endemic areas. Phytohemagglutinin (PHA) stimulated [3H]thymidine incorporation in MNC in all groups except the arsenical skin cancer group. However, when a low concentration of As2O3 (2.5 x 10(-7) M) was added to PHA-stimulated MNC, a tremendous amplification of the uptake of [3H]thymidine was noticed in patients with arsenical skin cancer. In this study, this phenomenon did not occur in cancers not related to arsenic. This result shows that arsenical carcinomas are hyperreactive to its specific etiology--arsenic. Arsenic seems to play a role as a co-stimulant of PHA similar to interleukin-1.


Chemoprevention of skin cancer in xeroderma pigmentosum. Xeroderma pigmentosum is a rare recessive disease with sun sensitivity, increased freckling and defective DNA repair. Xeroderma pigmentosum patients have more than a 1000-fold increased risk of developing skin cancer including basal cell carcinoma, squamous cell carcinoma and melanoma. We studied chemoprevention of new skin cancers with oral retinoids in xeroderma pigmentosum patients who had multiple skin cancers. Xeroderma pigmentosum patients were cleared of all pre-existing tumors surgically and then treated with high dose (2 mg/kg/day) oral isotretinoin (13-cis retinoic acid, Accutane) for two years and then for one year off treatment. Patients were examined at regular intervals for new tumor formation and for side effects. Five xeroderma pigmentosum patients had a total of 121 basal or squamous cell carcinomas in 2 years before treatment and only 25 tumors during 2 years of treatment. The tumor frequency increased 8.5-fold after the drug was discontinued (New Engl J Med 318: 1633-1637, 1988). Toxicity (cutaneous, triglyceride, liver-function or skeletal abnormalities) prompted subsequent use of a low dose protocol. Patients were treated initially with 0.5 mg/kg/day oral isotretinoin and the dose was increased sequentially to 1.0 or 1.5 mg/kg/day. We found that toxicity was less with the lower doses. The lowest effective, least toxic dose varied among the xeroderma pigmentosum patients.


Skin-infiltrating lymphocytes in normal and disordered skin: activation signals and functional roles in psoriasis and mycosis fungoides-type cutaneous T cell lymphoma. T lymphocytes recruited into the skin can experience several different outcomes. On the one hand, they may be recruited by adhesion molecules and chemoattractants to enter the perivascular space, but never undergo activation. Other T cells undergo activation and further differentiation under the influence of the cutaneous milieu. These activated lymphocytes then coordinate specific and non-specific immune responses characteristic of inflamed tissue. We have explored two models for studying the activation and function of skin infiltrating T lymphocytes (SIL's). In the first model, we have identified a family of Langerhans cell-related professional dendritic antigen presenting cells that exist in the epidermis and dermis of normal skin, atopic skin, and mycosis fungoides skin. These have APC abilities to activate freshly recruited resting blood T cells that are distinct from another family of macrophage-related cells abnormally present in sunburned or psoriatic skin. In the second model, we examined the function of cells that have already been recruited into the skin of patients with psoriasis and mycosis fungoides. Lesional psoriasis and mycosis fungoides T cells exhibited a variety of T cell receptor gene rearrangements, conclusively demonstrating that heterogeneous populations of T lymphocytes exist in inflamed human skin. From psoriasis, clones were identified that were particularly effective at inducing normal keratinocytes to assume "psoriatic" phenotypic features and functions. Thus, lesional psoriatic SIL's could induce HLA-DR, ICAM, and CDw60 on normal keratinocytes. In addition, psoriatic SIL's induced increased keratinocyte proliferation and cytokine profile changes characteristic of psoriatic epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)


Structure of desmoplakin and its association with intermediate filaments. Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)


Epidermolysis bullosa and the search for the basis of hereditary disorders of the skin. During the past two years the application of newer strategies of molecular genetics has resulted in the identification of genes whose mutations underlie five diseases of the skin. This is likely to be but the start of an unprecedented expansion in our understanding of these disorders and, most likely, of our ability to treat them.


A model to study the fate of genetically-marked keratinocytes in culture. In this study we demonstrate a method for analyzing the spatial distribution or fate of progeny keratinocytes derived from single progenitor cells. The method relies upon the use of retroviral vectors to introduce a reporter gene into replicating cells and to effect integration and expression of that new gene. All progeny cells from that initial cell inherit and express the transferred gene. The reporter gene is the E. coli beta-galactosidase gene (B-gal), which encodes a histochemically-detectable product in the cytoplasm. Using this method, we show that foci of genetically marked, B-gal positive cells can be readily identified in submerged cultures and we term this grouping of cells a "clonal proliferation unit". Analysis of B-gal stained whole mounts and paraffin sections allows visualization of the proliferative potential and differentiating capacity of clonogenic cells. This model will allow exploration of how agents known to alter epidermal proliferation and differentiation affect lineage relationships.


5-S-cysteinyldopa in urine and tumors.

5-S-cysteinyldopa in urine and tumors. 1) The urinary 5-S-CD contents in malignant melanoma subjects (n = 135) and non-melanoma subjects (n = 204) were measured by HPLC. These results suggest that, as a biochemical marker, periodic measurement of urinary 5-S-CD is quite useful for evaluating the determinations of stage classification (UICC, 1987), and the detection of metastases, the therapeutic efficacy of operation or immunochemotherapy against malignant melanoma. 2) Quantitative analyses of 5-S-CD values in tissues from primary malignant melanoma lesions (n = 24) and pigmentary tumors other than melanomas (n = 136) showed 80.6-821.4 ng/mg and N.D.-55.0 ng/mg respectively. In view of the above findings, it was suggested that the pigmentary tumors can be diagnosed as malignant melanoma if the 5-S-CD value in the tissues is higher than 100 ng/mg.


Serum 5-S-cysteinyldopa (5-S-CD) as a marker of melanoma progression. We previously reported that serum 5-S-cysteinyldopa (5-S-CD) tended to elevate earlier and reflect melanoma progression better than urinary 5-S-CD. In patients without metastatic melanomas, serum concentration and urinary excretion of 5-S-CD and 6-hydroxy-5-methoxy-indole-2-carboxylic acid (6H5MI2C) were within the upper limits of normal controls. In this report, we presented more precisely the changes in these melanin-related markers and clinical courses of four melanoma patients. Serum and 24-hour urine samples were serially collected and assayed every 1 to 4 months. Three of them developed stage IV malignant melanomas and died of metastatic disease. 6H5MI2C in serum and urine did not reflect the progression of disease. Among the 4 parameters considered, 5-S-CD in serum appeared to be the best biochemical marker for melanoma progression. Serum 5-S-CD over the upper limit of 10 nmol/L was suggested as a serious sign of the progression of melanoma.


Gangliosides of melanoma.

Gangliosides of melanoma. The relationships between the ganglioside composition of melanomas and their biologic behavior were investigated. (1) The amount of GM2 and/or GD2 in melanoma cells injected into nude mice correlated with the tumor growth rate. (2) GD2 content of melanoma cell lines correlated with sensitivity to radiation and vincristine. (3) GM2 expression of melanoma cells correlated with sensitivity to lymphokine-activated killer cells. (4) Gangliosides inhibited the proliferation of human T cells stimulated with interleukin-4 or interleukin-2. Based on these results, we proposed a hypothesis for the role of melanoma-associated gangliosides in the biologic behavior of melanomas and suggested a prospective melanoma treatment related to the gangliosides.