Precipitin Bands Research Articles

900: A REPORT OF DISSEMINATED HISTOPLASMOSIS IN AN IMMUNOCOMPROMISED PATIENT FOLLOWING COVID-19 INFECTION

Introduction: Histoplasmosis results from spore inhalation of Histoplasma capsulatum, an endemic fungus in the soil of the Ohio and Mississippi River Valleys of the United States. While the majority of infections with H. capsulatum are asymptomatic and self-limited, immunosuppressed patients may develop severe, disseminated disease. Few reports have described disseminated histoplasmosis following SARS-CoV-2 (COVID-19) infection such as described in this case. Description: A 69-year-old female was evaluated for shortness of breath. Her medical history was significant for rheumatoid arthritis on methotrexate and recent COVID-19 infection treated with dexamethasone. Initial CT-angiography of the chest showed patchy bilateral ground glass opacities. She had leukopenia with white blood cell count of 1.3 K/uL and elevated procalcitonin of 5.88 ng/mL. Broad spectrum empiric antibiotics were initiated. On day 6, the patient decompensated and required ICU transfer. Urine histoplasma antigen, M and H precipitin bands, and serum galactomannan antigen returned positive. Amphotericin B was initiated. The patient underwent bronchoscopy with bronchoalveolar lavage showing atypical reactive macrophages with rare intracellular organisms suspicious for fungi. Repeat CT of the chest, abdomen, and pelvis showed extensive bilateral ground glass opacities, bilateral pulmonary calcifications consistent with granulomas, and calcifications in the spleen and liver. On hospital day 10, the patient required intubation due to hypoxemic respiratory failure. Despite appropriate antifungal therapy, she had worsening encephalopathy, leukocytosis, erythema nodosum, and renal failure. She passed away on day 19. Discussion: This case highlights the development of disseminated histoplasmosis in an immunocompromised adult following COVID-19 infection. It was not until treatment with high dose glucocorticoids that the patient developed disseminated disease and declined. It is important to be aware of the impact that the COVID-19 pandemic has on the emergence of infectious disease and the role its treatment plays in immune suppression. Disseminated histoplasmosis is a consideration for immunocompromised patients in high risk, H. capsulatum endemic areas who are being treated for COVID-19 with high doses of steroids for prolonged periods.

Mediastinal Mass with Unusual Etiology.

Mediastinal Mass with Unusual Etiology.

Skin Hypersensitivity and Precipitating Antibodies Against Allergic Bronchopulmonary Aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is a very fatal disease and more than four million people have been affected. We collected data of forty-five patients and a skin hypersensitivity test was performed. We also evaluated precipitating antibodies against Aspergillus fumigatus. Instant hypersensitivity reactions were recorded in 22.22% of patients and 6.66% displayed delayed type III reaction. 4.44% of patients exhibited precipitin bands and three infections of ABPA among 45 infected people. A literature survey was also made to understand Aspergillus fumigatus.

HYPERSENSITIVITY PNEUMONITIS DUE TO AN OUTDOOR ANTIGEN IN THE HOME

Introduction Common etiologies of hypersensitivity pneumonitis (HP) include avian, bacterial, fungal, occupational, and agricultural exposures. We present a case of HP to an external avian antigen that became present in the home. Case description A 69-year-old non-smoking retired teacher with adult-onset asthma presented with an 18-month history of dry cough without improvement with ICS/LABA or H2 antagonists. She had been treated with prednisone 10mg daily for four months with difficulty tapering due to cough. She denied fevers or weight loss. She had no pets although previously had a bird feeder in her yard but denied pigeon exposure. Exam was notable for diffuse rhonchi. Chest CT showed patchy ground glass densities. Spirometry demonstrated FEV1 1.70 (74%), FVC 2.41 (80%), FEV1/FVC 0.71. An initial HP panel was negative, but IgG sent later for pigeon droppings was positive. Due to high suspicion for HP, patient's home furnace filter was analyzed which showed a precipitin band to pigeon droppings. After changing home filters and avoiding pigeons, her cough resolved, and prednisone was tapered to 1mg. Discussion Organic material from pigeon droppings outdoors can become aerosolized and compromise indoor air quality. The diagnosis of chronic HP may be difficult to confer in patients with persistent asthma. Our patient's refractory cough, CT findings, serum IgG and filter pigeon precipitins, and symptom resolution with avoidance met criteria for HP. Despite not owning or working with birds and a negative commercial lab HP panel, a high suspicion allowed for identification of an unsuspecting outdoor source within the home.

Assessment of the neutralizing potency of antisera raised against native and γ-irradiated Naja nigricollis (black-necked spitting cobra) venom in rabbits, concerning its cardiotoxic effect.

The present study was designed to prepare a specific safe antiserum for Naja nigricollis using γ-irradiated (1.5KGy and3KGy) venoms. Rabbits were used for active immunization using irradiated venoms (1.5 and 3 kGy) as a toxoid, mice were used for determination of LD50 post immunization and the rats were used for neutralization of the cardiotoxic effect of venom. Results of the immunodiffusion test indicated that the sera of rabbits raised against non-irradiated, 1.5- and 3-kGy γ-irradiated venom, had the same results of precipitin bands. A significant inhibition of phospholipase A2 activities was obtained when neutralized with native, γ-irradiated (1.5KGy and3KGy) venoms. On the other hand, preincubation of the venom ½ LD50 (0.154 mg/kg i.p.) with each antiserum (non-irradiated or irradiated venom) at 37°C for 1 h in a ratio (1:4) produced a significant reduction in the values of creatine kinase and creatine kinase isoenzyme-MB. However, significant elevation in aspartate aminotransferase level and no change in lactate dehydrogenase level were observed. So the results of this study indicated that the irradiated venom treatment reduces the cardiotoxic effect of venom in immunized immunization animals for preparing vaccines.

Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin-Darby bovine kidney cell line.

Aim:Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test.Materials and Methods:Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test.Results:Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test.Conclusion:BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.

Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

Phaeohyphomycosis byAlternaria alternarnata: A Case study:In-vitroSensitivity and Exoantigen Studies.

Alternaria alternata is the most frequently encountered species of Alternaria causing opportunistic mycoses. Two cases of phaeohyphomycosis are being reported in which Alternaria alternata was isolated from peripheral blood samples of 35 year and 21 year old male TB and post operative patients respectively. The direct microscopic examination of the blood samples revealed dark dematiaceous, septate mycelium. A. alternata was repeatedly isolated from the clinical samples of both the patients. Ketoconazole with the MIC of 10 mgml−1 was found to be the best effective drug against both the Alternaria alternata strains tested in vitro. In the present study we tried to evaluate the role of exoantigens in the serological diagnosis of such infections. The exoantigen of A. alternate on SDS-PAGE analysis exhibited two bands of 15Kda and 67Kda. The antisera raised in rabbit against. A alternata exoantigen showed humoral response after one week of immunization of ODD method. No cross reactivity was seen with antisera raised in rabbit against Curvularia verreculosa and Cladosporum cladosporiodides. Formation of specific precipitin bands and no cross reactivity suggests the usefulness of Alternaria alternata exoantigen as diagnostic tool. The antisera analysis by SDS-PAGE revealed several additional bands particularly higher than 67 Kda molecular weight as compared to the normal (unchallenged) sera from the same rabbit. Whether any of these bands could be treated as specific marker for diagnosis needs further investigations.

Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results.

Purification and Characterization of Cytotoxin Produced by a Clinical Isolate of Vibrio cholerae O54 TV113

Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 μg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40-80 % ammonium sulfate precipitation, gradient-anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.

Identification of some viruses causing mosaic on lettuce and characterization of Lettuce mosaic virus from Tehran Province in Iran

Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in lettuce fields in Tehran province. In this study, 452 infected lettuce plants having viral infection symptoms including, mosaic, mottling, leaf distortion, stunting defective heading, were collected from the fields throughout Tehran province. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus(CMV), Tomato spotted wilt virus (TSWV) and Arabis mosaic virus (ArMV) were determined with DAS-ELISA. LMV, CMV and TSWV were found on lettuce in this region, but no infection by ArMV was found. Percentage of single infection by LMV, CMV or TSWV was 21, 16 and 10% respectively. Also, 16% of samples were co-infected with LMV+CMV, 8% with LMV+TSWV and 8% with CMV+TSWV. 5% of samples were infected to all of these viruses. LMV was found in 49%, CMV in 44% and TSWV in 31% of samples totally. Therefore, LMV is major agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and the first report of CMV and LMV in Tehran province. Three LMV infected samples were collected and their characteristics were determined. After mechanical inoculation, these isolates produced symptoms on Chenopodium quinoa, Chenopodium amaranticolor, Gomphrena globosa, Nicotiana benthamiana, Lactuca sativa cv. Mantilia and cv. Terocadero (which contains the mo11 resistance gene and susceptible respectively), but not cv. Salinas 88 (which contains the mo12 resistance gene). LMV was purified and LMV polyclonal antiserum was produced in rabbit by a series of intravenous and intramuscular injections, the titre of this antiserum was 1:1024. Gel double diffusion test (GDDT) was performed, and precipitin bands appeared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting showed the presence of coat protein 29 kDa. In Immunocapture reverse transcription PCR (IC-RT-PCR) with a LMV specific primer pair, an aproximately 1300 bp fragment was amplified. Key words: Lettuce mosaic virus, Cucumber mosaic virus, Tomato spotted wilt virus, Immunocapture reverse transcription PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Modulation of the Pharmacological and Biochemical Actions of Leiurus quinquestriatus (L.q) Scorpion Venom by Exposure to Gamma Radiation

Back ground This study was undertaken to evaluate the effect of gamma radiation (1.5 KGy & 3 KGy) on L.q scorpion venom. This was carried out by studying the toxicological, biochemical & immunological properties of the venom before and after exposure to gamma radiation. Material and methods Animals, venom, antivenin, gamma radiation, 125I. Results Data revealed that the toxicity of irradiated venom (1.5 KGy & 3 KGy) decreased as compared to that of the native one. LD50 of irradiated venom were 3.5 mg/kg & 7.5 mg/kg respectively while, that of the native venom was (0.39 mg/kg). Moreover, the distribution of 125I-labeled L.q venom was studied in male Swiss mice tissue using chloramine-T method by being injected intravenously. At various time intervals, urine and blood were collected and the animals were sacrificed. Brain, lungs, heart, liver, kidneys, spleen, intestine, bone and muscle were isolated in order to determine the radioactivity content. The highest contents of 125I-labeled L.q venom were found in the liver and kidney that were quickly excreted into the urinary tract. Trial to label irradiated (1.5 & 3 KGy) L.q venom was unsuccessful due to its decomposition. For that reason the utilization of the labeled irradiated L.q venom is of no meaning. In immunodiffusion technique, both irradiated and native venoms exhibited prominent precipitin bands indicating high concentration of specific antibodies against polyvalent antivenin with venom. Furthermore, the effect of half the LD50 of native or irradiated (1.5 KGy) L.q venom was studied on the activities of certain enzymes: ASAT, ALAT, LDH, CPK, CPK-MB, and the levels of total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and the oxidative stress biomarkers (plasma MDA & blood GSH). Biochemical measurements showed that scorpion venom envenomation caused significant (p < 0.05) elevation in serum ASAT, ALAT, LDH, CPK as well as CPK-MB activities, blood GSH level while, caused significant (p < 0.05) reduction in triglyceride, HDL-cholesterol. In contrast, the 1.5 KGy gamma irradiated L.q venom showed no alterations except in HDL-cholesterol that significantly decreased compared to that of the non-envenomated normal rats. Conclusion These results indicated that 1.5 KGy gamma irradiation of venom offer an effective method for reducing the toxic effect of venom.

Modulation of the Pharmacological and Biochemical Actions of Leiurus quinquestriatus (L.q) Scorpion Venom by Exposure to Gamma Radiation

Back ground This study was undertaken to evaluate the effect of gamma radiation (1.5 KGy & 3 KGy) on L.q scorpion venom. This was carried out by studying the toxicological, biochemical & immunological properties of the venom before and after exposure to gamma radiation. Material and methods Animals, venom, antivenin, gamma radiation, 125I. Results Data revealed that the toxicity of irradiated venom (1.5 KGy & 3 KGy) decreased as compared to that of the native one. LD50 of irradiated venom were 3.5 mg/kg & 7.5 mg/kg respectively while, that of the native venom was (0.39 mg/kg). Moreover, the distribution of 125I-labeled L.q venom was studied in male Swiss mice tissue using chloramine-T method by being injected intravenously. At various time intervals, urine and blood were collected and the animals were sacrificed. Brain, lungs, heart, liver, kidneys, spleen, intestine, bone and muscle were isolated in order to determine the radioactivity content. The highest contents of 125I-labeled L.q venom were found in the liver and kidney that were quickly excreted into the urinary tract. Trial to label irradiated (1.5 & 3 KGy) L.q venom was unsuccessful due to its decomposition. For that reason the utilization of the labeled irradiated L.q venom is of no meaning. In immunodiffusion technique, both irradiated and native venoms exhibited prominent precipitin bands indicating high concentration of specific antibodies against polyvalent antivenin with venom. Furthermore, the effect of half the LD50 of native or irradiated (1.5 KGy) L.q venom was studied on the activities of certain enzymes: ASAT, ALAT, LDH, CPK, CPK-MB, and the levels of total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and the oxidative stress biomarkers (plasma MDA & blood GSH). Biochemical measurements showed that scorpion venom envenomation caused significant (p < 0.05) elevation in serum ASAT, ALAT, LDH, CPK as well as CPK-MB activities, blood GSH level while, caused significant (p < 0.05) reduction in triglyceride, HDL-cholesterol. In contrast, the 1.5 KGy gamma irradiated L.q venom showed no alterations except in HDL-cholesterol that significantly decreased compared to that of the non-envenomated normal rats. Conclusion These results indicated that 1.5 KGy gamma irradiation of venom offer an effective method for reducing the toxic effect of venom.

Comparative study between the protective effects of Saudi and Egyptian antivenoms, alone or in combination with ion channel modulators, against deleterious actions of Leiurus quinquestriatus scorpion venom

This study compared efficacy of two polyvalent antivenoms (Saudi Arabian and Egyptian), against lethality and pathophysiological changes of Leiurus quinquestriatus quinquestriatus ( LQQ) scorpion venom in mice. Additionally, the study examined whether treatment with selected ion channel modulators, lidocaine, nimodipine or amiodarone would be effective, alone or combined with the antivenoms. The protein concentration of the Saudi antivenom was 1/3 of Egyptian, indicating lesser immunogenicity, while both preservative contents were within limits. In immunodiffusion experiments, both exhibited prominent precipitin bands indicating high concentrations of specific antibodies. Neutralizing capacities (60–70 LD 50) stated on labels were confirmed. Both antivenoms significantly ( P < 0.001) prolonged survival time (from 26.9 ± 1.18 min, 100% dead with venom to 224–300 min, 0–30% dead) of envenomed mice, whether injected iv before or 5 min after venom. Injection of either antivenom plus ion channel modulators, gave comparable results to that observed in mice treated with antivenoms alone. The Na + channel blocker lidocaine and the Ca 2+ channel blocker nimodipine on their own significantly protected the animals ( P < 0.05), but to a lesser extent. The two antivenoms, significantly ameliorated the venom-evoked changes in serum LDH ( P < 0.001) and CKMB ( P < 0.01) plus cardiac TNFα and nitrate/nitrite levels ( P < 0.001). When combined with lidocaine or nimodipine, the effects were not greater than antivenom alone. Moreover, the antivenoms ameliorated characteristic venom-evoked changes in the isolated perfused Langendorff hearts. Lidocaine and amiodarone were more effective than nimodipine. In Conclusion both Saudi and Egyptian antivenoms protected mice from the pathological and lethal effects of LQQ scorpion. Sodium and calcium channel blockers, lidocaine and nimodipine, may be useful when antivenoms are not available.

Chemical and immunological differentiation of exoantigens from four Bipolaris sorokiniana strains

Four strains (3, 5, 8, and 18) of Bipolaris sorokiniana were assessed as to their antigenic and partial chemical composition through antisera produced in rabbits. The antisera were immunochemically analyzed by double diffusion and immunoelectrophoresis. The partial chemical characterization of exoantigens was obtained by measuring the amount of proteins, carbohydrates and lipids in the strains. Immunoelectrophoresis showed that strain 18 antigens stimulated the production of nine precipitin bands. No significant differences were found between the antisera produced by the animals immunized with the same antigen. Partial chemical analysis showed protein content between 173.4 and 332.2 μg mL −1; carbohydrate levels between 0 and 64.3 μg mL −1; and lipid content from 0 to 166 μg mL −1. Immunodiffusion and immunoelectrophoresis associated with chemical characterization are relevant when phenotypic traits of different strains of the same fungus species are studied.

Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly.

We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [(35)S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Ability of Gamma-Irradiated Polyvalent Antivenin to Neutralize the Toxicity of the Egyptian Cobra (Naja haje) Venom

In this study, the lethality as well as the biochemical and histological effects of Cobra (Naja haje) envenomation at a sublethal dose has been investigated in mice and rats. The venom injected intraperitoneally in rats (0.2 mg/kg) produced marked increase in the activities of ALT, AST, ALP and LDH. Also, serum glucose, urea and creatinine levels were significantly elevated. These results are in accordance with the histological findings of the liver that showed vacuolated hepatocytes and scattered necrotic and haemorrhagic areas together with congestion and dilatation of blood vessels and sinusoids. The spleen also displayed small diffuse white pulps with ill-defined outlines and extravasation of blood that extended to infiltrate the widened sinusoids. The venom also produced severe degeneration of the cardiac muscle with loss of striations and extensive haemorrhage inbetween the myocardial bundles. These biochemical and histological envenomation disorders, were markedly and effectively neutralized by the polyantivenins (non-irradiated and gamma-irradiated in a sterilizing dose of 25 kGy). The neutralization ability of both antivenins was the same against a lethal dose of the venom (2.5 LD50). The minimal protective dose of the polyantivenins was calculated to be 16000 μg/20 gm mouse. Rats injected with mixtures of venom and antivenins (non-irradiated and irradiated) showed a decrease in all elevated biochemical parameters investigated. The histological examination also showed less severe injuries in all organs examined (liver, spleen and heart) which appeared more or less normal with very few abnormalities remaining. Comparative study was also done on these antivenins using immunodiffusion technique which showed the same precipitin bands with the tested venom. Thus, it can be concluded that both antivenins (non irradiated and 25 KGy gamma-irradiated) have similar immunoglobulins and have no differences in their antilethal and enzymatic neutralizing ability as well as in ameliorating the degree of tissue damage induced by the venom.

Invasive aspergillosis in cancer.

Ten confirmed cases of invasive aspergillosis (IA) in cancer patients were analysed retrospectively. Eight were pulmonary, one was sinonasal and one was cutaneous. The majority of patients had haematological malignancies (7), the remaining three were cases of solid tumours. Fever was present in all 10 cases. Cough and lung signs were present in all eight cases of invasive pulmonary aspergillosis. Haemoptysis was encountered in three of nine cases of pulmonary and sinonasal aspergillosis. Mortality was low (2%). While corticosteroids, antibiotics and anticancer chemotherapy/radiotherapy were factors predisposing the patients to IA, neutropenia was perhaps responsible for their mortality. Seven of the patients had other associated pathogens isolated in culture in addition to Aspergillus spp. Aspergillus fumigatus was the predominant species, followed by A. flavus, A. glaucus, A. nidulans and A. niger. Direct microscopic examination (in six of seven cases) and culture (six of seven cases) correlated well with radiographic and clinical findings in cases with lung involvement. Serology for anti-Aspergillus antibodies performed by gel diffusion precipitin test was positive in one case of sinonasal aspergillosis, wherein only one precipitin band was observed. Correlation of clinical symptoms, consistent radiographic findings and microbiological work-up (the latter including a triad of direct microscopy, culture and serology) are required to arrive at a diagnosis of IA, especially where histology cannot form the mainstay of diagnosis.

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells.

A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10-30 microg/ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 microg/ml could induce mRNA accumulation of tumour necrosis factor-alpha, interleukin (IL)-1beta and IL-6 as well as iNOS. In contrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components--one that reacts specificially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce inflammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.