Isolation and characterization of a new human microsporidian, Encephalitozoon hellem (n. sp.), from three AIDS patients with keratoconjunctivitis

A new human microsporidian was isolated from cultures of Madin-Darby canine kidney cells incubated with conjunctival scrapings or corneal tissues from three AIDS patients with keratoconjunctivitis. The three isolates were morphologically similar to Encephalitozoon cuniculi. The spores averaged 1 x 1.5-2.0 microns, had six to eight polar filament coils, displayed monokaryotic nuclei, and possessed relatively thick endospores with irregularly shaped exospores. Organisms developed within a parasitophorous vacuole. By SDS-PAGE analysis, the three isolates appeared to be identical but were different from E. cuniculi. Identical banding patterns on Western blots of the three isolates were expressed by each patient's serum. By Western immunoblotting, murine antisera to E. cuniculi reacted to several antigens of the new AIDS-related microsporidian, whereas murine antisera bound weakly to Nosema corneum. The name Encephalitozoon hellem (n. sp.) is proposed to identify this new human microsporidian.     

Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors.

The DNA genome of a human papillomavirus (HPV), tentatively designated HPV-EV, was molecularly cloned from hand to leg lesions of a patient with epidermodysplasia verruciformis, a chronic skin disease associated with a 30% risk of developing cancer. Using stringent hybridization conditions, we observed less than 5% homology between HPV-EV and the cloned genomes of HPV-1, HPV-4, HPV-5, and HPV-5a. HPV-EV DNA showed approximately 6% homology with HPV-2 and 36% homology with HPV-3. These data suggest that HPV-EV is partially related to HPV-3. Using 32P-labeled cloned HPV-EV as probe in Southern blot hybridization experiments, we detected HPV-EV-related DNA in the carcinoma in situ (Bowenoid lesion) of the vulva of the patient from which HPV-EV was isolated. HPV-EV-related DNA was detected in 2 of 10 vulva carcinomas and in 2 of 31 cervical carcinomas. Related DNA sequences were found in papillomas from each of two patients with condyloma acuminata (anogenital warts), which is of interest considering that condylomas have been reported to convert occasionally to carcinomas. The positive vulva DNAs were also probed with other cloned HPV DNAs: HPV-1, HPV-4, and HPV-5a-related sequences were not detected; HPV-3 and HPV-2 DNA probes detected strong and weak DNA bands, respectively, of the same size as found with HPV-EV. The HPV DNA sequences were present in the positive tumors mainly as free viral DNA molecules; no evidence for integration into cellular DNA was found. The emerging biological picture with papillomaviruses is that cells transformed by these viruses are maintained in a transformed state by free episomal genomes. Thus, our findings are consistent with the idea, but by no means establish, that HPVs play a role in human cancer by a similar mechanism.     

Isolation of Chlamydia pneumoniae from a patient with acute bronchitis]

Chlamydia was isolated from the throat of a 15-year-old male patient with acute bronchitis. The Chlamydia isolate, YK-41, was stained with FITC-conjugated monoclonal antibodies specific to C. pneumoniae and the genus Chlamydia, whereas staining of monoclonal antibody specific to C. trachomatis was negative. These results indicated that the strain YK-41 could be identified as C. pneumoniae. Serum IgM antibody against C. pneumoniae was detected in high titer in the patient in the acute phase using the microimmunofluorescence (MIF) test, serum IgG antibody against C. pneumoniae demonstrated a fourfold antibody titer rise between acute and convalescent serum using MIF test. Thus, our patient almost certainly contracted acute bronchitis caused by C. pneumoniae.     

Isolation of cytomegalovirus from synovial cells of a patient with rheumatoid arthritis

Synovial cells from the joint of a patient with rheumatoid arthritis (RA) showed foci of cytopathic effects during their 3rd subculture in vitro. Electron microscopy of these cells revealed findings characteristic for cytomegalovirus (CMV), and immunofluorescent staining using antiserum specific for CMV was positive. The patient had classical seropositive RA, was not taking immunosuppressive agents, and had no clinical manifestations of CMV disease at the time of synovectomy 3 months earlier. Titers of antibodies to CMV were studied in the sera and synovial fluids of 3 additional patients with RA, and in 1, elevated IgM antibodies to CMV were found, indicating recent infection. Additional studies for CMV in RA are indicated.     

Isolation of Exophiala dermatitidis from pigmented sputum in a cystic fibrosis patient

A 16‐year‐old female with cystic fibrosis (CF) presented with an acute respiratory exacerbation during which black flecks were observed in the spontaneously expectorated sputum. The production of this pigmented sputum was subsequently attributed to Exophiala dermatitidis hyphae. Treatment with antibiotics, corticosteroids, and antifungal medications led to an initial resolution of symptoms and clearance of the black pigment from her sputum. However, the patient again presented nine months later with reappearance of the pigmented flecks and concomitant clinical deterioration and was subsequently treated with an extended course of voriconazole. To the authors' knowledge, this is the first case report of fungal colonization by E. dermatitidis presenting as black flecks spontaneously expectorated in CF sputum. Pediatr Pulmonol. 2010; 45:508–510. © 2010 Wiley‐Liss, Inc.     

Isolation of a neuropeptide-degrading carboxypeptidase from the human stomach.

The aim of this investigation was to isolate and characterize a neuropeptide-degrading carboxypeptidase from the muscular and mucosal layers of the human stomach. The carboxypeptidase was solubilized from membrane preparations of gastric muscle and mucosa using Triton X-100. The detergent-solubilized enzyme was purified to apparent electrophoretic homogeneity by affinity chromatography using lisinopril or potato carboxypeptidase inhibitor as an affinity ligand. The enzyme had an apparent molecular weight of 34,300 and was bound by concanavalin A and is thus a glycoprotein. The carboxypeptidase removed C-terminal leucine, phenylalanine, or tryosine residues from peptides including angiotensin I, [Leu5]enkephalin, kinetensin, neuromedin N, neurotensin, and xenopsin. It had an alkaline pH optimum and was inhibited by lisinopril, potato carboxypeptidase inhibitor, ethylenediaminetetraacetic acid, 1,10-phenanthroline, and 8-hydroxyquinoline. Immunoblotting indicated that the gastric carboxypeptidase cross-reacted with an antibody raised against a carboxypeptidase isolated from mast cells of human skin. The gastric carboxypeptidase released from gastric mast cells upon degranulation may act to degrade and inactivate neuropeptides in the stomach wall.     

Isolation of megakaryocytes from human placentae

Shedding of cytoplasm from circulating megakaryocytes (MKs) within the pulmonary vasculature suggests the lungs are an important site for normal platelet production. Fetal lungs receive only a minor fraction of the circulating blood volume. The placenta may act as a site for intrauterine platelet formation. Isolation of MKs from fetal vessels within the placenta has not been previously reported. Immediately after delivery, 3 human placentae were subjected to forward and retrograde perfusion across the placental capillary bed on the fetal side. MKs in perfusates were harvested by 'whole blood filtration' and identified by morphological and immunochemical methods. All perfusates yielded MKs. Qualitatively MKs with copious cytoplasm were more commonly found in perfusates collected from fetal arteries compared with those from fetal veins. This is consistent with filtration of MKs and fragmentation of their cytoplasm within the placental microcirculation to produce platelets. Perfusion of human placentae followed by filtration of perfusates is a useful technique for harvesting fetal MKs and permitting further elucidation of their physiological role.     

Isolation of Bordetella pertussis in blood culture from a patient with multiple myeloma

Bordetella pertussis is a fastidious aerobic, Gram-negative coccobacillus that causes the classical disease of whooping cough. We present a 63 years old man with multiple myeloma who was admitted to the medical department with cough, hoarseness and fever. Bronchopneumonia was suspected and two courses of beta-lactam antibiotics had beneficial effect on his clinical condition, but his respiratory symptoms persisted. Eventually, B. pertussis grew in an aerobic blood culture. Delayed treatment with clarithromycin had little or no effect on his cough. To our knowledge this is the third reported case of B. pertussis bacteraemia. All three patients have been immunocompromized. Growth in blood culture may be slow, and prolonged incubation (at least 6 days) of the blood culture bottles and subculture to a special culture medium is necessary for isolation of B. pertussis.     

Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.     

Isolation of Listeria monocytogenes from a patient with sealed ruptured thoracoabdominal aortic aneurysm

We are reporting a case of sealed rupture of thoracoabdominal aortic aneurysm associated with the isolation of Listeria monocytogenes. The patient was a 75-year-old man with previous history of hypertension that had not required medication for the 3 years prior to hospital admission. He was admitted due to chest pain, but he was afebrile. There were no clinical findings indicating infection, although CRP was slightly elevated. During his clinical course, a sealed rupture of a thoracoabdominal aortic aneurysm was found and replaced with an artificial artery. After surgery, he was treated for 2 weeks with sultamicillin. He was discharged from hospital on the 43rd postoperative day. No bacteria were observed after microscopic examination of gram stained samples from the thrombus that was present in the sealed rupture of the aneurysm. However, a L. monocytogenes strain isolated from the thrombus only after enrichment culturing by HK medium at 37 degrees C for 4 days. By histopathology, there was a slight cellular infiltration of lymphocytes and neutrophils at the aperture of the aneurysm. Although L. monocytogenes strains possess major pathogenic genes, such as prfA, hlyA, plcA, plcB, mpl, inlA, inlB and actA that can be identified by PCR, none of the evidence indicated that this case was a mycotic aortic aneurysm due to L. monocytogenes.     

病人血清中登革病毒的分离与鉴定

目的应用细胞分离法从登革热病人急性期血清中分离登革热病毒并应用免疫荧光法证实病毒的血清型。C6/36细胞长成单层后接种急性期血清,以5~7天为一代,观察细胞病变;感染后的细胞使用型特异性抗体进行免疫荧光实验证实其血清型。在10份急性期病人血清中,有6份标本成功分离出登革热I型病毒,其结果和RT-PCR等实验方法的结果一致,分离率为60%。     

Isolation of human T-cell leukemia virus type I from a transformed T- cell line derived spontaneously from lymphocytes of a seronegative Egyptian patient with mycosis fungoides

Mycosis fungoides (MF) is a rare form of cutaneous T-cell lymphoma that may be associated with human T-cell leukemia virus type I (HTLV-I) infection. Using the polymerase chain reaction, the HTLV-I pX region was constantly detected in the genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of an HTLV-I antibody-seronegative Egyptian MF patient enrolled in a study to isolate HTLV-I from North Africa. A CD4+ and interleukin-2 (IL-2) receptor-positive T-cell line was established when the phytohemagglutinin-stimulated PBMCs of that patient were maintained in IL-2-containing culture medium. The cell line (EMF) was initially IL-2 dependent and then became IL-2 independent after gradual withdrawal of the IL-2. The cells reacted positively with monoclonal antibodies specific for the HTLV-I Env or HTLV-I Gag proteins. Using the Southern blot analysis, HTLV-I provirus could be detected in the genomic DNA extracted from the EMF cells. Limited nucleotide sequence of the env region showed more than 95% homology between the EMF provirus and other known HTLV-I isolates. Western blot analysis of the cell lysates showed the expression of the HTLV-I structural proteins. These data imply that a transforming HTLV-I provirus may be present, at least in certain cases of MF, regardless of the presence or absence of the specific antibodies.     

Isolation of a novel endogenous opiate analgesic from human blood.

Based upon its ability to inhibit opiate receptor binding, a low-molecular-weight substance (600) has been isolated from human plasma by extraction into butanol and ion exchange, molecular sieve, and thin-layer chromatography. When this substance, termed anodynin, is microinjected into rat periaqueductal gray matter, it causes a profound, long-lasting analgesia which is prevented by prior injection of the opiate antagonist naloxone. Anodynin (opiate receptor binding material) levels in serum from hypophysectomized rats are less than 5% of values obtained in sham-operated controls. Anodynin differs from enkephalin, a morphine-like peptide isolated from brain, in its sensitivity to enzymatic loss of opiate receptor inhibitory potency, thin-layer chromatographic mobility, and behavioral effects. Anodynin might be a hormone that acts on peripheral opiate receptors in the classical manner, but might also, due to its lipophilic nature and small size, penetrate into the brain to produce centrally mediated behavioral effects.