Survival of cultured allografts in patients with burns assessed with probe specific for Y chromosome.

The aim of the study was to determine the fate of cultured skin allografts in patients with burns. In situ DNA hybridisation with a Y probe (pHY 2.1) was used to detect cells carrying the Y chromosome (the probe being visualised by the alkaline phosphatase-antialkaline phosphatase method) in biopsy specimens taken from cultured allografts derived from donors of the opposite sex to the recipients (20 patients with burns). Specimens were taken within a week, between one and three weeks, between four and six weeks, and more than six weeks after grafting. Only two of the 27 biopsy specimens contained cells that were the same sex as the donor; both were taken within a week after grafting. In the 25 other specimens the epithelial cells were the same sex as the recipient. Cultured skin allografts showed no evidence of survival in patients with burns, which suggests that they are probably not suitable for long term management of burns but may be useful as short term biological dressings.     

Long-term survival of human skin allografts in patients with immunosuppression

Severe burn patients lack adequate skin donor sites to resurface their burn wounds. Patients with severe burn injuries to areas such as an entire face are presently reconstructed with skin grafts that are inferior to normal facial skin. This study was designed in part to determine whether human skin allografts would survive, repopulate, and persist on patients with immunosuppression and after discontinuation of immunosuppression. Small split-thickness skin grafts were synchronously transplanted at the time of renal transplantation from six renal transplant donors to recipients. All six patients were immunosuppressed with the usual doses of renal transplant immunosuppressants (methylprednisolone, cyclosporine, prednisone, and azathioprine). The skin allografts were biopsied when rejection was suspected and at various intervals. Special histologic studies were performed on skin biopsy specimens. Class II DNA tissue typing was performed on transplanted and autogenous skin biopsy specimens of four patients. Fluorescent in situ hybridization was performed successfully on skin biopsies of four patients' transplanted skin and on two of these four patients' autogenous skin. All six human skin allografts sustained a 100 percent take and long-term clinical survival. DNA tissue typing performed on skin allograft biopsy specimens from patients taking immunosuppressants all revealed donor and recipient cells. DNA tissue typing performed on autogenous skin biopsies from the same patients all revealed only recipient cells. Fluorescent in situ hybridization performed on allograft and autogenous specimens from patients taking immunosuppressants revealed transplanted donor cells with rare recipient cells in the allograft and only recipient cells in the autogenous skin. This study of six patients proves that it is possible for human skin allografts to survive indefinitely on patients taking the usual dosages of immunosuppressants used for renal transplantation. There was minimal repopulation of skin allografts by autogenous keratinocytes and fibroblast while patients were taking immunosuppressants. Immunosuppression was discontinued in two patients after renal transplant rejection after 6 weeks and 5 years. When immunosuppression was discontinued after 5 years in one patient, the skin allograft cells were destroyed and replaced with autogenous cells, but the skin graft did not reject acutely and persisted clinically. It is hypothesized that the acellular portion of the skin allograft was not rejected acutely because of relatively low antigenicity and because it acted as a lattice for autogenous cells to migrate into and replace rejected allograft skin cells. No chimerism was seen in autogenous skin in the skin-renal transplant patients in this study.     

Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome.

To determine the survival of cultured allogeneic keratinocytes transplanted to a deep dermal bed 24 tattoos that had been removed by deep shave excision in 19 patients were grafted with sheets of cultured allogeneic keratinocytes from donors of the opposite sex. Cells carrying the Y chromosome were identified in biopsy specimens taken from the graft site by in situ DNA hybridisation with a biotinylated Y probe (pHY 2.1) and visualised with a technique using immunoperoxidase. The cultured allograft sites were biopsied one, two, and three weeks after transplantation. No male cells were identified in any biopsy specimen from female patients who were given transplants of male cultured keratinocytes, and all biopsy specimens from male patients, who received female cultured keratinocytes, showed percentages of male cells within the normal range for male skin. The beneficial effects of cultivated allogeneic keratinocytes result from effects on wound healing other than forming a successful graft that "takes."     

High-performance liquid chromatography of lipids for the identification of human metabolic disease.

We describe a system for quantitative lipid analysis employing ternary gradient high-performance liquid chromatography with evaporative light scattering detection. This technique was applied to extracts of cultured fibroblasts, cultured lymphocytes, and leukocytes and to liver and spleen biopsy specimens. Separation of nonpolar lipids, glycolipids, phospholipids, and sphingolipids was achieved in a single run. Detection did not depend on the presence of any specific chemical reactions, uv absorption, or fluorescence. The sensitivity of the technique is well below 200 ng for individual lipids, and many individual lipid classes were detected in samples as small as 1 mg of total protein, the yield of a single flask of cultured skin fibroblasts. The characteristic stored lipids cholesterol ester and sphingomyelin were seen in excess in human fibroblast cultures from patients with Wolman's disease and Niemann-Pick disease, respectively. A biopsy spleen sample from a patient with Gaucher's disease showed a large glucosylceramide peak. This system provides a tool for detecting lipids that accumulate in tissues of patients with currently unidentified metabolic storage disorders.     

Mycobacterium tuberculosis DNA in tissue affected by sarcoidosis.

OBJECTIVE--To investigate the prevalence of Mycobacterium tuberculosis DNA in granulomatous tissues from patients with sarcoidosis and from controls matched for age, sex, and tissue by using the polymerase chain reaction. DESIGN--Single blind control trial. SUBJECTS--16 patients with sarcoidosis who had undergone diagnostic biopsy of lung, skin, or lymph node and 16 patients with squamous cell carcinoma or Hodgkin''s disease to act as controls. In addition, four lung specimens infected with M tuberculosis were included as positive controls. RESULTS--M tuberculosis DNA was present in sarcoid tissues containing granulomas from seven of the 16 patients and one of the 16 matched controls. Two of the four specimens known to be infected with M tuberculosis were positive in the controlled experiment. CONCLUSION--These figures suggest that M tuberculosis DNA is detected as readily in patients with sarcoidosis as in patients with frankly tuberculous tissues and imply that M tuberculosis may be linked to the cause of sarcoidosis.     

Infection of the skin by Mycobacterium marinum: report of five cases.

Five patients 6 to 47 years of age had bacteriologically confirmed Mycobacterium marinum infections of the skin. In four patients the initial lesions were chancriform and were on a finger or a hand; ascending lymphadenitis followed. The other patient had nodular inflammatory lesions on one cheek. Biopsies were performed in four patients; all specimens showed a granuloma, and acid-fast bacilli were identified in one of the four. Four of the five patients were untreated; the fifth was given antituberculous drugs. In all five patients the condition was chronic, with progressive resolution of the lesions during follow-up periods of 1 to 6 years. M. marinum infections of the skin are rare in Canada. This may be due in part of lack of clinical awareness or failure to identify the organism. Biopsy specimens should be obtained under sterile conditions and nondigested material should be inoculated into the footpads of mice.     

Small bowel biopsy for malabsorption: comparison of the diagnostic adequacy of endoscopic forceps and capsule biopsy specimens.

Biopsy specimens of the small bowel were obtained from 40 patients suspected of having malabsorption. Four different techniques were used at a single session--namely, endoscopic biopsy of the descending duodenum using paediatric and standard size forceps and suction capsule biopsy of the descending duodenum and the proximal jejunum. Specimens were compared for size, adequacy, and ability to confirm or exclude mucosal abnormality. Fourteen patients had villous atrophy. In all patients four biopsy specimens were obtained with paediatric endoscopic forceps and four with standard endoscopic forceps. No capsule biopsy specimen was retrieved from the duodenum in three patients and from the jejunum in five patients. Specimens were considered to be adequate in 36 patients when paediatric forceps were used, in 39 when standard forceps were used, in 28 on duodenal capsule biopsy, and in 32 on jejunal capsule biopsy. This study indicates that the most reliable method for diagnosing or excluding villous atrophy is endoscopic forceps biopsy of the descending duodenum, provided that at least four specimens are obtained with standard size forceps.     

Eradicating Helicobacter pylori and symptoms of non-ulcer dyspepsia.

OBJECTIVE--To examine the effect of eradication of Helicobacter pylori on symptoms of non-ulcer dyspepsia. DESIGN--Four week prospective study. SETTING--One hospital outpatient and endoscopy department. PATIENTS--90 adults with persistent symptoms typical of non-ulcer dyspepsia but no clinical or endoscopic evidence of other peptic, biliary, pancreatic, or malignant disease; all had histological and microbiological evidence of infection with H pylori. 83 patients completed the treatment regimen. INTERVENTION--Colloidal bismuth subcitrate 120 mg four times a day for four weeks (27 patients); metronidazole 400 mg and amoxycillin 500 mg each three times a day for one week (27); and bismuth subcitrate 120 mg four times a day for four weeks, metronidazole 400 mg three times a day for one week, plus amoxycillin 500 mg three times a day for the first week (29). MAIN OUTCOME MEASURES--Change in symptom scores determined with questionnaire; histological evidence of gastritis and microbiological evidence of presence of H pylori in biopsy specimens. RESULTS--Overall, H pylori was eradicated in 41 (49%) patients. Although gastritis scores improved significantly in only patients in whom H pylori had been eradicated (from 1.56 to 0.61, p less than 0.01 v from 1.83 to 1.07, p = 0.52) mean symptom scores after treatment were similar in patients in whom H pylori had or had not been eradicated (3.0 v 2.3, NS). Similarly the mean symptom score improved whether or not gastritis improved (2.8 v 3.1 respectively, p = 0.72). The observations were similar for treatment groups analysed individually. CONCLUSION--Antral infection with the organism does not seem to have an important aetiological role in non-ulcer dyspepsia short term.     

Chemosensitivity testing in a tumor acquisition, propagation, and preservation program

Chemosensitivity assays were carried out as part of a tumor acquisition, propagation, and preservation program for cancer biotherapy. In addition to biopsy specimens, tumor cells propagated in culture or tumor xenografts grown in nude mice were submitted for chemosensitivity assay when sufficient biopsy material was unavailable. Chemosensitivity was tested utilizing the adhesive tumor cell culture system. A total of 154 specimens was submitted for testing; 96 specimens were assayed. Success rates were 55% for primary cancer biopsies, 67% for metastases, 69% for xenografts, and 70% for cell lines. There were no significant differences evident when the sensitivity to drugs of tumor cells originating from biopsies, xenografts, or tissue culture were compared. Sufficient data were available for 18 patients to compare clinical results of drug treatment with predictive results from the chemo-sensitivity assay. Assay results indicating insensitivity appeared to predict resistance; however, assays indicating sensitivity were not predictive. These results suggest that propagated tumor material, such as xenografts and cultured cell lines, may be useful when biopsy tissue is not available.     

Colonic spirochetosis of colony-raised rhesus macaques associated with Brachyspira and Helicobacter

Colonic spirochetosis is an inflammatory bowel disease that affects a broad range of hosts, including human and non-human primates. The disease in humans and non-human primates is characterized by intimate attachment of the anaerobic spirochetes Brachyspira aalborgi and B. pilosicoli, and some unclassified flagellated microbes along the apical membrane of colonic enterocytes. Although the presence of spiral-shaped bacteria with single polar flagella and blunted ends in colonic spirochetosis is well established, the identities of many of these organisms is still unknown. Recently, Helicobacter species with a morphology similar to the flagellated bacteria present in colonic spirochetosis have been cultured from intestinal specimens obtained from rhesus macaques, some with idiopathic colitis. The purpose of the present study was to determine whether or not the flagellated bacteria seen in the colons of rhesus macaques with colonic spirochetosis are Helicobacter. The presence of flagellated bacteria alone (n=2) or together with spirochetes (n=1) in formalin-fixed and paraffin-embedded colons of three rhesus macaques with the naturally occurring disease was demonstrated by immunohistochemical staining and ultrastructural examination. Total DNA extracted from affected and control intestinal specimens was amplified by polymerase chain reaction (PCR) using Helicobacter 16S rRNA gene-specific primers. Comparative nucleotide sequence analysis of PCR products cloned from positive reactions indicated that two distinct Helicobacter genomospecies were present either alone or in combination with Brachyspira in the colons of rhesus macaques with microscopic lesions indicative of colonic spirochetosis.     

Physiological diversity of rumen spirochetes.

Bovine rumen fluid contained relatively large numbers of spirochetes capable of fermenting polymers commonly present in plant materials. Polymers such as xylan, pectin, and arabinogalactan served as fermentable substrates for the spirochetes, whereas cellulose did not. Furthermore, spirochetes cultured from rumen fluid utilized as growth substrates hydrolysis products of plant polymers (e.g., D-xylose, L-arabinose, D-galacturonic acid, D-glucuronic acid, cellobiose), but did not ferment amino acids. Viable cell counts of spirochetes capable of fermenting individual plant polymers or their hydrolysis products yielded minimum values ranging from 0.2 X 10(6) to 4 X 10(6) cells per ml of rumen fluid. Thirteen strains of rumen spirochetes were characterized in terms of their fermentation products from glucose, the guanine plus cytosine content of their DNA, their ultrastructure, and their ability to ferment pectin, starch, or arabinogalactan. Of the 13 strains, 6 fermented glucose mainly to formate, acetate, and succinate, whereas the remaining 7 strains did not produce succinate, but instead formed ethanol, in addition to formate and acetate. The succinate-forming strains had two periplasmic (axial) fibrils per cell, measured 0.2 to 0.3 by 5 to 8 micrograms, had a guanine plus cytosine content of the DNA ranging from 36 to 38 mol%, and lacked the ability to ferment pectin, starch, or arabinogalactan. The ethanol-forming strains had from 8 to more than 32 periplasmic fibrils per cell, tended to be larger in cell size than the succinate-forming strains, and had a guanine plus cytosine content of the DNA ranging from 41 to 54 mol%. Some of the ethanol-forming strains fermented pectin, starch, or arabinogalactan. The results of this study indicate that the bovine rumen is inhabited by a physiologically and morphologically diverse population of spirochetes. It is likely that these spirochetes contribute significantly to the degradation of plant materials ingested by the ruminants.     

EXPERIENCES WITH ACTH AND CORTISONE IN SELECTED DERMATOSES

Fifty patients with various kinds of skin diseases who were not adequately relieved by conventional therapy were treated with ACTH or cortisone given systemically.Almost all patients with disseminated neurodermatitis had dramatic initial response, but in only about half the cases was improvement maintained when use of the drugs was discontinued.It appeared that in other skin diseases, such as lupus erythematosus, scleroderma, psoriasis, dermatomyositis and pemphigus, while improvement may be noted for a time, relapse to the original state occurs after the treatment is stopped.In four cases of chronic discoid lupus erythematosus, although some improvement was observed when steroid therapy was given, the histologic pattern of biopsy material taken from the lesions after treatment still was characteristic of the disease.     

Enzyme activities of Lyme disease and relapsing fever borreliae

Activities of glycosidases ( n = 8), esterases ( n = 10), arylamidases ( n = 63), acid phosphatase, alkaline phosphatase and phosphoamidase were tested in 47 Borrelia strains. Forty-four were B. burgdorferi strains; 22 of which were isolated from human specimens (skin 13, cerebrospinal fluid six, and one each from blood, heart muscle and synovia), 13 were isolated from various organs of laboratory animals infected via tick bite or with human isolates, and nine from ticks. The remaining three were the relapsing fever strains B. coriaceae , B. hermsii , and B. turicatae. Strains were of low and high passage but the number of subcultures did not influence the enzyme patterns obtained by utilization of chromogenic substrates of constitutive enzymes. Glycosidase activity was absent in almost all strains tested. Esterase activity was high on molecules of chain length 9 carbons. 2-Naphthylamide derivatives were utilized by strains of human, rodent or tick origin in a range of 66.6 to 83.1%. Almost all strains utilized substrates for acid and alkaline phosphatase and phosphoamidase. Chymotrypsin activity was only found in three and two strains from specimens of human and rodent origin, respectively; and γ-glutamyltransferase activity only in three human skin isolates. No strain tested displayed trypsin activity. Overall, the specific activities of constitutive enzymes of the Borrelia strains tested are widely similar. Thus, the enzyme profiles did not discriminate between human, animal and tick isolates, or between human isolates of Borrelia whether cultivated from cerebrospinal fluid or from skin biopsy of Lyme borreliosis patients.     

Experimental exposure of Canadian toads to Basidiobolus ranarum

Experimental transmission of the fungus Basidiobolus ranarum was induced in two treatment groups of Canadian toads (Bufo hemiophrys) and caused a fatal mycotic dermatitis. Seven of 10 (70%) toads that had their ventral skin mildly abraded and exposed to B. ranarum developed hyperemia, and sloughing of their ventral skin and died. Toads with abraded ventral skin or exposure to infected skin also were affected statistically at a higher rate than those with abraded skin and exposure to pure cultures of B. ranarum inoculated into their water source. Of toads showing clinical disease, B. ranarum was identified by both impression smears and histology in all cases, but not from toads that appeared clinically healthy. The organism was cultured from 5 of 7 (71%) toads with clinical disease but not from any toad that appeared clinically healthy (n = 28). This study documents methods of experimental transmission of B. ranarum, an organism responsible for causing a mycotic dermatitis that is fatal to toads.     

Percutaneous fine-needle aspiration biopsy of intra-abdominal masses.

Percutaneous fine-needle aspiration biopsies were performed in 51 patients with various intra-abdominal masses localized by palpation, radiologic studies, ultrasonography or radioisotope scanning. Biopsy specimens were considered positive for malignant disease in 35 (85%) of the 41 patients with such disease, including 26 (96%) of the 27 with metastases. There was one false-positive diagnosis of malignant disease from the biopsy specimens. Surgery became unnecessary as a result of aspiration biopsy in at least 12 patients. One patient showed evidence of intrahepatic bleeding during liver biopsy but recovered spontaneously, and the liver appeared normal at laparotomy 3 weeks later. Aspiration biopsy is an accurate, relatively painless, inexpensive and safe method of establishing a diagnosis of intraabdominal malignant disease. Considerable experience of the cytologist is necessary for good results.     

Utilization of electron microscopy in the prenatal diagnosis of genetic disease.

The use of electron microscopy as a further method of diagnosis of disease in cultured skin fibroblasts and cultured amniotic fluid fibroblasts is presented. It was demonstrated that Tay-Sachs disease, Fabry's disease, and metachromatic leukodystrophy had distinctive abnormalities in both cultured skin fibroblasts and cultured amniotic fluid fibroblasts. It was shown that control of culturing conditions made it possible to distinguish normal cell lines from certain cell lines carrying known genetic diseases.     

Treatment of Nelson's syndrome by pituitary implantation of yttrium-90 or gold-198.

Eight patients with Nelson''s syndrome were treated with a pituitary implant of yttrium-90 or gold-198 four to 16 years after adrenal surgery. All had considerable pigmentation. One already had cranial nerve abnormalities and visual field defects and had had both a craniotomy and deep x-ray treatment. Radiographs showed that the pituitary fossa was abnormal in seven patients. A biopsy performed in six cases showed mucoid (or basophil) adenoma in all. In the four specimens examined ACTH was identified by electron microscopy or immunofluorescence, or both. Patients were followed up after pituitary implantation for three months to 12 years. All showed decreased pigmentation, and six became normal. Four patients regained normal ACTH levels and the other two studied had decreased levels. In no case did new cranial nerve disease or further sellar expansion develop since operation, and two patients showed remodelling of the sella. Complications were temporary leakage of cerebrospinal fluid and diabetes insipidus in one patient and gonadotrophin deficiency in another.     

The ratio of lipidperoxides to superoxide dismutase activity in the skin lesions of patients with severe skin diseases: an accurate prognostic indicator

We studied 35 patients with active inflammatory skin diseases, measuring the levels of lipidperoxides and of the oxygen radical scavenging enzyme superoxide dismutase (SOD) in biopsy specimens of skin lesions. Lipidperoxide levels were markedly elevated in all patients. In fifteen patients with disease that was severe and highly resistant to therapy, SOD activity was only slightly increased, in comparison with normal controls. In contrast, in the twenty patients with mild disease that responded well to therapy, SOD activity was markedly elevated. The ratio of lipidperoxide levels to SOD activity was thus an accurate prognostic indicator, being elevated only in the group not responding to treatment. These findings suggest that the severity of allergic inflammatory skin disease and/or the response to treatment may in part be governed by the degree to which the patient's SOD activity is up-regulated in response to the generation of tissue-damaging substances such as lipidperoxides. Interestingly, our studies revealed the SOD activities of both normal and inflamed skin to be unexpectedly high; our data suggest that SOD plays a critical role in protecting the skin from the effects of oxygen radicals and ultraviolet light.     

The generation of enzymatically active plasmin on the surface of spirochetes

The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to the host by a feeding Ixodid tick. The spirochete subsequently disseminates through the skin, enters the bloodstream, and becomes systemic. A potential mechanism for this invasiveness was identified with the discovery that B. burgdorferi can bind components of the plasminogen activation system (PAS). The methodology for analyzing the generation of enzymatically active plasmin on the surface of this organism is given, and applied to measure spirochete viability, strain differences, and breakdown of extracellular matrix (ECM) macromolecules. Plasmin acquisition by B. burgdorferi was measured photometrically by a specific chromogenic substrate. The growth of B. burgdorferi in culture was not affected by the presence of active plasmin on the spirochete surface. Plasmin-coated B. burgdorferi degraded the purified (ECM) components fibronectin, laminin, and vitronectin, but not collagen. The addition of B. burgdorferi with surface plasmin to a radiolabeled, native ECM resulted in degradation of noncollagenous protein, as measured by release of solubilized radioactivity. Breakdown of purified ECM components or native ECM did not occur after exposure to untreated spirochetes or spirochetes treated with uPA or PLG alone. These results provide in vitro evidence that enzymatically active plasmin on the surface of B. burgdorferi may be partially responsible for its invasiveness.     

1982至2020年中国肾移植受者合并隐球菌病的系统分析

隐球菌病是威胁肾移植患者生命的严重感染性疾病,本文旨在报道我国肾移植患者合并隐球菌病（cryptococcosis in kidney transplant patients,C-KT）的情况。通过对208例患者的资料进行分析,发现隐球菌病平均发病时间为肾移植后(5.48±4.09)年,就诊时间为发病后(27.28±30.69)d,入院后确诊时间为(31.6±44.0)d。本研究中C-KT患者误诊率为23.1%,误诊患者占所有死亡患者的37.5%。此病可侵袭全身各个系统,最常见于中枢神经系统（脑）、呼吸系统（肺）和皮肤等。临床特征除发热、头痛、呕吐外,还常见为恶心、咳嗽、胸闷气短,结节、红斑、脓肿溃疡伴或不伴皮肤疼痛、触痛。诊断方法主要是墨汁染色、乳胶凝集素试验等,检出标本主要为脑脊液,血液,肺泡灌洗液,肺、皮肤病例活检组织等。首选治疗药物为两性霉素B脂质体、氟康唑、5-氟尿嘧啶和伏立康唑等,临床中有氟康唑耐药病例的报道。C-KT患者的总死亡率为15.8%。本文对全面了解肾移植患者合并隐球菌病的特征及对本病的诊断与治疗具有重要的意义。