Pathological and immunological protection induced by inactivated reverse genetics-based H3N8 equine influenza vaccine candidate in murine model.

Equine influenza (EI) is an important viral respiratory disease of equines caused by influenza A virus (IAV). The antigenic drift in IAVs necessitates regular updating and harmonization of vaccine strain with the circulating virus. The reverse genetics-based recombinant viruses could be easy instrument in generating vaccine against circulating virus in a quick and effective manner. Present study has been envisaged to evaluate the immunogenicity and protective efficacy of inactivated recombinant equine influenza virus (rgEIV) vaccine candidate having six segments from H1N1 virus (A/WSN/33/H1N1) and HA (hemaglutinin) and NA (neuraminidase) segments from H3N8 equine influenza virus [(A/eq/Jammu-Katra/06/08) of clade 2 of Florida sublineage] generated through reverse genetic engineering. BALB/c mice were immunized with inactivated rgEIV adjuvanted with aluminium hydroxide gel and challenged with H3N8 virus (A/eq/Jammu-Katra/06/08). The protective efficacy was evaluated through serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry and residual virus quantification. Immunizations induced robust humoral immune response as estimated through hemagglutination inhibition assay (HAI). The antibodies were isotyped and the predominant subclass was IgG1. The vaccine candidate produced mixed Th1 and Th2 responses through stimulation of IFN-γ, IL-2, IL-4 and IL-6 expression. Immunization protected mice against challenge as reflected through reduction in clinical signs and body weight loss, early recovery, mild pathological changes (gross and histopathological lesions) as evident through scoring of lesions, low residual virus in nasopharynx and lungs quantified through egg titration and quantitative reverse transcriptase PCR (qRT-PCR). The study demonstrates that inactivated recombinant EIV generated through reverse genetic approach provides equivalent protection to that observed with inactivated whole H3N8 EIV vaccine. Keywords: equine influenza; reverse genetics; vaccine; pathology; murine model.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Hydrogen dependent surface morphology study of plasma deposited SiN(x):H films for two gas systems SiH4/NH3 and SiH4/N2.

Partially amorphous silicon nitride thin films were deposited using plasma enhanced chemical vapor deposition technique using the two gas systems: SiH4/NH3 and SiH4/N2. Fourier Transform infrared spectroscopy was employed to derive the relative changes in the bonded hydrogen content with increasing flow rates of NH3 and N2. Surface morphology was monitored using atomic force microscopy. Root mean square surface roughness was found to be dependent on the NH3 and N2 flow rates, unlike silicon nitride films deposited by rf magnetron sputtering with variation in (N2/Ar) (Li et al. Thin Solid Films 334 (1998) 140). The discrepancy has been explained in the light of bonded hydrogen content in these films. The X-ray diffraction technique has also been used to observe the phases of the nitride films which showed the presence of silicon nitride grains oriented in (200), (400) and (221) directions in the predominantly amorphous as-deposited SiN(x):H films.

Etiological prevalence and antifungal sensitivity patterns of dermatophytosis in India - A multicentric study.

BACKGROUND: The prevalence of dermatophytes varies with season, geographical area, socio-economic factors and effective management strategies. AIMS: The aim of the study was to assess the prevalence of pathogenic dermatophytes, clinical types of dermatophyte fungal infection, and in vitro antifungal drug susceptibility testing against dermatophytes. METHODS: Three hundred and ninety five patients with dermatophytosis were enrolled from five cities (Mumbai, Delhi, Lucknow, Kolkata and Hyderabad) across India. All patients were subjected to clinical examination and investigations, including potassium hydroxide microscopy, fungal culture and antifungal drug susceptibility testing. RESULTS: Trichophyton rubrum was the most common species identified (68.4%), followed by T. mentagrophytes (29.3%). Within species, T. mentagrophytes was prevalent in humid environmental conditions (Mumbai and Kolkata), whereas T. rubrum was prevalent in noncoastal areas (Delhi, Lucknow and Hyderabad). Tinea corporis (71.4%) and tinea cruris (62.0%) were the common clinical types observed. antifungal drug susceptibility testing data indicated that minimum inhibitory concentration required to inhibit the growth of 90% of organisms (MIC-90) was lowest for griseofulvin (0.25-3.0 µg/mL). Among oral antifungals, the mean MIC of itraconazole was within the range (0.84 [0.252] µg/ mL), whereas high mean MIC values were reported for terbinafine (0.05 [0.043] µg/mL). Among topical agents, lowest mean MIC values were reported for luliconazole (0.29 [0.286] µg/mL), eberconazole (0.32 [0.251]) µg/mL and amorolfine (0.60 [0.306]) µg/mL. LIMITATIONS: Lack of correlation between in vitro antifungal susceptibility and clinical outcome and absence of defined MIC breakpoints. CONCLUSION: T. rubrum was the most common, followed by T. mentagrophytes as an emerging/codominant fungal isolate in India. Tinea corporis was the most common clinical type of dermatophytosis. Mean MIC of terbinafine was above the reference range, while it was within the range for itraconazole; griseofulvin had the lowest mean MIC. Luliconazole presented the lowest mean MIC values across cities.

Evaluation of Vitreous Substitutes in Managing Complicated Vitreo-retinal Surgeries.

BACKGROUND: Vitreous substitutes presently in use for intraoperative tamponade are perfluorocarbon liquids (PFCL) and for post operative tamponade are silicon oil (SO), sulphur hexafluoride (SF6) and perfluorocarbon gas (PFC). Several factors are important for a thorough understanding of each of these vitreous substitutes. The absorption or necessity for removal, indications for use (including clinical studies and special surgical methods), additives and complications of use have to be considered. METHODS: Three port standard pars plana vitrectomy was performed in 60 consecutive cases requiring intra-vitreal intervention. PFCL was used intra-operatively in 30 cases (PFCL group and X group). The eyes were implanted with SO (silicon oil subgroup), PFC (C3F8 sub-group) and SF6 (SF6 sub-group) in twenty cases each. Apart from best corrected visual acuity (BCVA), the retinal status and the longevity of gas when used, changes and reaction in both the anterior and posterior segments were noted. RESULT: The difference of postoperative BCVA in the PFCL and non-PFCL groups was statistically significant with p < 0.001. In both the PFCL and non-PFCL groups the difference between preoperative and postoperative BCVA was statistically significant with p < 0.0001. All three vitreous substitutes studied are effective given the case where they had been utilised. CONCLUSION: PFCL is invaluable in the management of complicated retinal detachment (RD). Where a vitreous microsurgery is indicated, the visual outcome is good. SF6 is useful for short-term tamponade. Silicon oil and C3F8 are useful for longer tamponade.

Semi-quantitative characterisation of ambient ultrafine aerosols resulting from emissions of coal fired power stations.

Emissions from coal fired power stations are known to be a significant anthropogenic source of fine atmospheric particles, both through direct primary emissions and secondary formation of sulfate and nitrate from emissions of gaseous precursors. However, there is relatively little information available in the literature regarding the contribution emissions make to the ambient aerosol, particularly in the ultrafine size range. In this study, the contribution of emissions to particles smaller than 0.3 mum in the ambient aerosol was examined at a sampling site 7 km from two large Australian coal fired power stations equipped with fabric filters. A novel approach was employed using conditional sampling based on sulfur dioxide (SO(2)) as an indicator species, and a relatively new sampler, the TSI Nanometer Aerosol Sampler. Samples were collected on transmission electron microscope (TEM) grids and examined using a combination of TEM imaging and energy dispersive X-ray (EDX) analysis for qualitative chemical analysis. The ultrafine aerosol in low SO(2) conditions was dominated by diesel soot from vehicle emissions, while significant quantities of particles, which were unstable under the electron beam, were observed in the high SO(2) samples. The behaviour of these particles was consistent with literature accounts of sulfate and nitrate species, believed to have been derived from precursor emissions from the power stations. A significant carbon peak was noted in the residues from the evaporated particles, suggesting that some secondary organic aerosol formation may also have been catalysed by these acid seed particles. No primary particulate material was observed in the minus 0.3 mum fraction. The results of this study indicate the contribution of species more commonly associated with gas to particle conversion may be more significant than expected, even close to source.

A rare cause of heart failure--primary hypoparathyroidism.

We report a patient who presented with congestive heart failure (ejection fraction 24.4%) and who had previous history of convulsions. Our investigations found him to be a case of primary hypoparathyroidism. He showed a dramatic response with the addition of calcium infusion therapy with almost full recovery of left ventricular function (67% ejection fraction after 16 days of the initial echo). We conclude that in a young patient a thorough investigation for heart failure is never complete without looking for endocrine and metabolic causes. The prognosis in these cases is much better, identification and treatment of the same will yield dramatic results.

Comparison of Phaconit Rollable IOL with Acrylic Foldable IOL.

BACKGROUND: Phaconit or ultra micro incision phacoemulsification cataract surgery involves phacoemulsification through a 0.9 millimetre sleeveless phaco tip and irrigating chopper followed by implantation of a rollable intraocular lens. The procedure leads to negligible astigmatism and faster visual recovery as compared to phacoemulsification with a foldable intraocular lens. METHODS: This prospective study analysed 80 cases of sub millimetre phaconit surgery with implantation of rollable intraocular lenses(IOL) in 40 cases and acrylic foldable IOL in the remaining 40 cases. Evaluation of efficacy and adaptability of procedure, equipment settings, operative constraints, postoperative complications, keratometric and topographic evaluation of induced astigmatism with visual outcome and patient's rehabilitation were studied. RESULTS: The intraoperative complications were surge/ chamber collapse in 16 (20%), iris chaffing in one and corneal burns in two cases. All cases had an induced astigmatism of less than or equal to ± 0.25 D in four to six weeks after rollable IOL and ± 0.5 D to ± 0.75 D after acrylic IOL implantation. All patients had best-corrected visual acuity of 6/6 by third post operative day. CONCLUSION: Phaconit with rollable IOL is a perfect blend of surgical skill, application of technology and ultra thin IOL.

The changing spectrum of severe falciparum malaria: a clinical study from Bikaner (northwest India).

BACKGROUND & OBJECTIVES: Recently there were reports from all over India about changing spectrum of clinical presentation of severe malaria. The present study was planned to study the same in the northwest India. METHODS: This prospective study was conducted on patients of severe malaria admitted in a classified malaria ward of a tertiary care hospital in Bikaner, Rajasthan (northwest India) during 1994 and 2001. It included adult patients of both sexes belonging to all age groups. The diagnosis of Plasmodium falciparum was confirmed by demonstrating asexual form of parasites in peripheral blood smear. All patients were treated with i.v./oral quinine. The specific complications were treated by standard WHO protocol. The data for individual complications for both the years were analysed by applying chi-square test. RESULTS: In a prospective study in 1994 the spectrum of complication was dominated by cerebral malaria (25.75%) followed by jaundice (11.47%), bleeding tendencies (9.59%), severe anaemia (5.83%), shock (5.26%), Acute respiratory distress syndrome-ARDS (3.01%), renal failure (2.07%) and hypoglycemia (2.07%) whereas in 2001 it was dominated by jaundice (58.85%) followed by severe anaemia (26.04%), bleeding tendencies (25.52%), shock (10.94%), cerebral malaria (10.94%), renal failure (6.25%), ARDS (2.08%) and hypoglycemia (1.56%). The sharp difference for presence of jaundice and severe anaemia in 2001 and cerebral malaria in 1994 was statistically significant. Similarly, the important cause of mortality in 2001 was multiple organ dysfunction syndrome (71.10%) with predominant presentation of jaundice and renal failure, whereas in 1994, it was cerebral malaria (77.96%). INTERPRETATION & CONCLUSION: The observation of changing spectrum of severe malaria in this study and a significant increase in presentation with jaundice as an important manifestation is highly essential for primary, secondary and tertiary level health care providers for proper diagnosis and management.

Safety and Efficacy of Sodium Bicarbonate versus Hyaluronidase in Peribulbar Anaesthesia.

BACKGROUND: 400 cases undergoing surgery for cataract under local anaesthesia were studied. Peribulbar anaesthesia involves injecting a mixture of 2% lignocaine, bupivicaine 0.5% and hyaluronidase into the peripheral space of the orbit through a single infero-lateral point. Sodium bicarbonate has been shown to reduce the time of onset of anaesthesia and pain perception when mixed with local anaesthetics. METHODS: This study compared two groups of patients (200 each), one receiving hyaluronidase mixed anaesthetic and the other sodium bicarbonate buffered anaesthetic. The groups were compared for effectiveness of the anaesthesia, its onset, duration and the final visual outcome. RESULTS AND CONCLUSION: Sodium bicarbonate was shown to reduce the time of onset and increase the successful block rate without any adverse affects.

Phacotrabeculectomy Versus Conventional Combined Technique in Coexisting Glaucoma and Cataract.

BACKGROUND: To evaluate and compare efficacy and outcome after single site phacotrabeculectomy and conventional combined surgery in cases of coexisting primary open angle glaucoma and cataract. METHODS: This prospective study on fifty patients of concurrent primary open angle glaucoma and cataract, who had undergone combined surgery as single site phacotrabeculectomy or conventional single site trabeculectomy with extracapsular lens extraction with IOL implantation in 25 cases each. Evaluation was based on operative and postoperative complications, control of IOP and visual outcome. The follow up period ranged between twelve months to eighteen months. RESULTS: The mean medically controlled preoperative intraocular pressure was 22 mm of Hg (Range 18 to 35 mm of Hg) by applanation method of tonometry. The range of postoperative intra-ocular pressure after one year was 11 to 22 mm of Hg in first and 14 to 26 mm Hg in second group. Failure to maintain optimum postoperative IOP without Beta-blocker was more frequent after conventional combined procedure. There was no significant difference in incidence and pattern of postoperative complications. CONCLUSION: Phacotrabeculectomy provides effective and sustained visual recovery and adequate control of intraocular pressure as compare to conventional combined procedure.

In vitro antibacterial effect of Withania somnifera root extract on Escherichia coli.

AIM: The aim was to investigate antibacterial activity of Withania somnifera (Ashwagandha), an Indian traditional medicinal plant against Escherichia coli O78, a pathogenic strain. MATERIALS AND METHODS: Two-fold serial dilutions of 20% aqueous W. somnifera root (WSR) extract were inoculated with E. coli O78 @ 1*10(7) colony forming units grown in nutrient broth. Following inoculation, turbidity optical density was measured by spectrophotometer at 600 nm in all the tubes at 0, 2, 4, 6 and 8 h of incubation at 37°C. RESULT: The results revealed that the maximum inhibition of bacterial growth was observed at 1:8 dilution of WSR extract. The highest dilution of the extract that showed inhibited growth of the test organism when compared with control was 1:16. Therefore, the minimum inhibitory concentration of aqueous extract of WSR is 1:16. CONCLUSION: It is concluded that WSR possessed good antibacterial activity, confirming the great potential of bioactive compounds and its rationalizing use in health care.

Potential radiosensitizing agents. 5. 2-Substituted benzimidazole derivatives.

A series of 2-substituted benzimidazoles and their derivatives have been synthesized and tested for their ability to selectively sensitize hypoxic Chinese hamster cells (V-79) toward the lethal effect of ionizing radiation. These compounds were prepared by reacting the 2-substituted benzimidazoles with 1,2-epoxy-3-methoxypropane in the presence of potassium carbonate. Reaction of the 2-nitro and 2-methylfonyl analogue with the epoxide also yielded a cyclized material, which was confirmed to be a benzimidazo[2,1-b]oxazole. In an attempt to increase the electron affinity, 5- or 6-nitro-2-substituted-benzimidazoles were also synthesized and then reacted with the epoxide to yield the corresponding 1-substituted derivatives. The results of the biological tests for the radiosensitizing activity of these agents against Chinese hamster cells (V-79) in culture indicated that the 2-nitro-substituted analogues were the most effective sensitizers in this series.

Potential radiosensitizing agents. 7. 4(5)-Iodo-5(4)-nitroimidazole derivatives.

A series of 4(5)-iodo-5(4)-nitro-1-substituted-imidazoles has been synthesized and tested for their ability to selectively radiosensitize hypoxic Chinese hamster cells (V-79) to the lethal effect of radiation. The reaction of 4(5)-iodo-5-(4)-nitroimidazole with 1,2-epoxy-3-methoxypropane and ethyl alpha-chloroacetate produced two isomeric products in each case, which were identified by their NMR spectra. The ethyl esters were further reacted with 3-picolylamine to produce corresponding amides. The 5-iodo-4-nitroimidazole-1-N-(3-picolyl)acetamide on further reaction with m-chloroperbenzoic acid produced the corresponding N-oxide. These compounds were generally more toxic to V-79 cells than the 2-nitroimidazole derivatives and were found to be more effective radiosensitizers in vitro. The 5-iodo-4-nitroimidazole derivatives were more efficient as sensitizers than the 4-iodo-5-nitroimidazole derivatives, and the sensitizing efficiency of this class of agents was found to have significant correlation with their partition coefficients.

Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of alpha and beta/beta' subunits of molecular weights 46,000 and 55,000/52,000, respectively. The ratio of alpha to beta/beta' subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The Km value of ATP was 19 +/- 1.3 microM, and the K0.5 values of calcium and calmodulin were 4.4 +/- 0.5 and 3.0 +/- 0.5 microM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the alpha-subunit but apparently not of the beta/beta' subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II alpha subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both alpha and beta subunits of hen brain CaM-kinase II cross-reacted with only the alpha subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II in several properties. Immunocross-reactivity suggested that there was similarity in the alpha but not the beta/beta' subunits of the hen brain enzyme and the brain enzyme of other species.

Enhanced mRNA expression of neurofilament subunits in the brain and spinal cord of diisopropyl phosphorofluoridate-treated hens.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, and a single injection of this compound (1.7 mg/kg, s.c.) produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. Clinically, the disease is marked by hindlimb ataxia followed by paralysis after some time. A characteristic feature of this neuropathy is axonal swelling in the initial stages and comparative dissolution of the accumulated material and degeneration of distal axons with disease progression. Axonal swelling consists of aggregated neurofilaments, microtubules, and proliferated smooth endoplasmic reticulum. We studied expression of neurofilament (NF) mRNAs in brain regions and spinal cord to elucidate their role in OPIDN. There was a 50-200% increase in NF transcripts in 24 hr after DFP administration. The NF-L mRNA level started falling after 1-5 days and came down to control level in susceptible brain regions (i.e. cerebellum and brainstem) and spinal cord, but not in cerebral cortex, which does not show degeneration of axons in OPIDN. Cerebral cortex exhibited elevated levels of both NF-L and NF-M transcripts in DFP-treated hens throughout the period of observation. The induction of NF messages is consistent with the previously reported effect on extension of neurites of human neuroblastoma cells in culture. The transient increase in NF messages in susceptible tissues either may be responsible for the delayed degeneration of axons in OPIDN or is the result of interruption of regulatory signal due to progressive degeneration of axons.

Induction of cytochrome P450 isozymes by simultaneous inhalation exposure of hens to n-hexane and methyl iso-butyl ketone (MiBK).

Chickens were exposed simultaneously to the industrial hexacarbon solvents n-hexane and methyl iso-butyl ketone (MiBK). n-Hexane has been shown to be neurotoxic in both humans and other vertebrates. While MiBK is not neurotoxic, it has been shown to greatly synergize the clinical appearance of neurotoxicity in animals exposed to both of these solvents. Groups of hens were exposed for 29 days in inhalation chambers to 1000 ppm n-hexane in combination with 10, 100, 250, 500, or 1000 ppm MiBK. Other groups received either 1000 ppm n-hexane, 1000 ppm MiBK, or ambient air and served as controls. A dose-dependent decrease in body weight and an increase in clinical effects were noted for the highest exposure groups (1000 ppm n-hexane combined with 1000, 500 or 250 ppm MiBK). There was an MiBK dose-dependent increase in cytochrome P450 content and benzphetamine N-demethylase activity, but there was no distinct pattern for ethoxyresorufin O-deethylase or cytochrome c reductase activities. Mixed-function oxidase levels and activities (cytochrome P450 content and benzphetamine N-demethylase) were elevated significantly (P less than 0.05) over controls even in the lowest MiBK group (10 ppm), although there were no clinical signs of neurotoxicity. Four different isozymes of cytochrome P450 were measured immunologically. There was a dose-dependent increase in three of the isozymes, two of which were phenobarbital inducible and one of which was induced by beta-napthoflavone. Quantitatively, the largest increase was in the PB-A isozyme, a phenobarbital-inducible isozyme which accounted for approximately 70% of the cytochrome P450 present in animals treated with MiBK. The results suggest that MiBK selectively induces cytochrome P450 isozymes leading to the metabolic activation of the weak neurotoxicant n-hexane to the potent neurotoxicant 2,5-hexanedione (2,5-HD).

Alteration in neurofilament axonal transport in the sciatic nerve of the diisopropyl phosphorofluoridate (DFP)-treated hen.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester that produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hens 7-14 days after a single s.c. dose of 1.7 mg/kg. In this study, hens were treated with a single dose of DFP (1.7 mg/kg, s.c.) 24 hr after [35S]methionine injection into the sacrolumbar region of their spinal cord, and killed 3, 7, 14, or 27 days post-DFP treatment. The rates of transport of labeled high (NF-H), medium (NF-M), and low (NF-L) molecular weight neurofilaments, and tubulin were faster in DFP-treated birds than in controls after 3 days. Subsequently, the rate of transport of these proteins started falling, so that the peaks of labeled proteins in control and DFP-treated hens were overlapping after 7 days. At 14 days, the peaks of NF-H, NF-M, and NF-L in treated hens were distinctly behind the corresponding peaks in control hens. This was again followed by an increase in transport of NF-H and NF-L, but not of NF-M, so that the labeled NF-H and NF-L showed the same pattern in control and treated hens after 27 days. The transient decrease in NF-H and NF-L axonal transport rate, and recovery correlated in a temporal manner with the previously reported increase of Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation of neurofilament proteins and inhibition of calpain activity in the sciatic nerve in OPIDN. Proteinase inhibition has been reported recently to result in enhanced phosphorylation of neurofilaments in some cells. The present study suggests that the enhanced phosphorylation of neurofilaments by DFP-increased Ca2+/calmodulin-dependent protein kinase activity may be contributing toward alteration in NF axonal transport and the development of OPIDN.

Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.

Highly purified Clara cells (93 +/- 3%) isolated from the lungs of rabbits were used to produce an antiserum against Clara cell secretory proteins. This antiserum was used to identify and study the biosynthesis and secretion of [35S]methionine-labeled proteins from isolated Clara cells. The antiserum recognized one major secretory protein with apparent molecular weight of 6 kDa and reacted weakly with a higher molecular weight protein of about 180 kDa. Biosynthesis and secretion of these proteins was not detected in preparations of isolated alveolar type II cells or alveolar macrophages. Immunocytochemical localization of the antigen with colloidal gold indicated a dual localization in bronchiolar Clara cells. Gold labeling was found over the osmiophilic secretory granules of Clara cells and smooth endoplasmic reticulum. In tracheal Clara cells, labeling was found mostly in association with secretory granules and relatively little in association with the smooth endoplasmic reticulum. Labeling was also found over the lamellar bodies of type II cells, although the reaction was weak. Labeling of ciliated cells, alveolar type I cells, capillary endothelial cells, and alveolar macrophages was not distinguishable from background. These data indicate that Clara cells of both the bronchioles and trachea of rabbits synthesize and secrete the low molecular weight protein previously called Clara cell secretory protein (CCSP). This antigen does not belong to that group of surfactant proteins whose molecular weights range from 26 to 40 kDa.