Increased culture recovery of Zygomycetes under physiologic temperature conditions

Poor recovery of Zygomycetes hyphae from tissue specimens may result from failure of current culture methods to mimic physiologic conditions found in hyphae-laden infected tissue. We describe the use of an in vitro model simulating Zygomycetes growth under necrotic or hypoxic tissue conditions. We preconditioned hyphae of clinical Zygomycetes isolates in flasks under anaerobic conditions using Ana-Packs (Becton Dickinson Microbiology Systems, Sparks, MD) at 37 degrees C for 48 hours, thus simulating in vivo growth in an infracted hypoxic lesion, and compared the recovery of paired inocula at 37 degrees C and 25 degrees C. Incubation of stock culture isolates at 37 degrees C resulted in significantly better culture recovery (about 10-fold) when compared with incubation at 25 degrees C (P < .0001). In addition, we similarly evaluated 25, 291 consecutive clinical specimens. Among 41 specimens, the yield of Zygomycetes cultures incubated at 37 degrees C (23/41 [56%]) was significantly higher than that incubated at 25 degrees C (9/41 [22%]; P = .0001). Overall, we found that culture recovery was significantly (254%) enhanced at 37 degrees C.     

Preparation of antiviral sera for immunofluorescence on infected tissue culture

Antisera were prepared in rabbits against parainfluenza types 1, 2, and 3, respiratory syncytial, herpes, and adenoviruses, either by intranasal administration or by injection of virus grown in RK₁₃ cells. These antisera gave satisfactory results when used for immunofluorescence by the indirect method on virus in tissue culture. No absorption of the antiviral serum was carried out.     

Ultrastructural and cytochemical changes in tissue culture infected with type 5 adenovirus

Summary Cytochemical and electron microscopic methods were used to study changes in the HEp-2 tissue culture infected with type 5 adenovirus. The infected tissue culture was noted to have a considerable decrease in succinate dehydrogenase activity in 24, 48 and 72 h. The activity of DPN- and TPN-diaphorases somewhat increased 10 h after infection and sharply decreased only on the 3rd day of investigation. The activity of acid and alkaline phosphatases did not change substantially within the first 48 h of infection and somewhat declined in 72 h. Non-simultaneous decrease in the activity of succinate dehydrogenase and DPN- and TPN-diaphorases was connected with the morphological characteristics of the localization. Since the virustiter considerably increased on the 8th day of investigation it is supposed that these enzymes are not decisive for virus reproduction. The infected tissue culture was noted to have a change in glycogen deposition manifested by lyoglycogen. Feulgen-positive inclusions, constantly encountered in infected tissue culture have a complex structure: they give an intensive reaction not only to DNA but also to total protein and sulfhydryl groups.(Presented by Acting Member of the Academy of Medical Sciences of USSR N. A. Maeveskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 61, No. 3, pp. 114–118, March, 1966     

Activation of C3 and binding to Aspergillus fumigatus conidia and hyphae

Complement activation by Aspergillus fumigatus may play a crucial role in stimulating binding and killing of this organism by phagocytes. We examined the amount and type of C3 deposited on resting conidia, swollen conidia, and hyphae of A. fumigatus after incubation in pooled human serum. All three life forms of A. fumigatus were potent activators of the complement cascade, with deposition on the organisms of similar amounts of C3 per unit of surface area. The rate of deposition was slowest for resting conidia, although maximal deposition was still achieved within 40 min. The roles of the alternative and classical pathways were assessed by use of serum chelated with magnesium EGTA [magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and with an alternative pathway reconstituted from the six purified alternative-pathway proteins. Complement activation by resting conidia was mediated by the alternative pathway. In contrast, there was a progressive dependence on the classical pathway as the fungal particles matured into swollen conidia and then hyphae. Treatment with hydroxylamine, which disrupts ester linkages, removed 89 to 95% of the C3 bound to all three forms of A. fumigatus. This released C3 contained a mixture of C3b and iC3b, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These data demonstrate that although all three forms of A. fumigatus are potent activators of the complement system, the transition from resting conidia to swollen conidia to hyphae results in progressive changes in the manner in which the fungal particles interact with the complement system. The lack of participation of the classical pathway in complement activation by resting conidia may have important implications regarding their ability to effectively stimulate phagocytes.     

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.     

Diffuse infiltration of Aspergillus hyphae in the thyroid gland with multinodular goiter

A 35-year-old woman presented with a thyroid mass, weakness and shortness of breath of 3 years duration. On physical examination, she had a diffusely enlarged thyroid gland with multiple nodules. There were no signs to suggest immune suppression. The patient farmed and raised livestock. Biochemical tests and hemogram were normal. She underwent surgery, and a histological examination of the surgical specimen revealed nodular hyperplasia. Microscopically, silver methenamine (PASM) stain-positive hyphae that divided into branches at 45° and conidia were detected beside the thyroid capsule, with conidia in the cystic nodule. Moreover, ischemic changes of the thyroid tissue were observed closer to the capsule. We report a case of Aspergillosis of the thyroid of a patient who underwent surgery for a multinodular goiter.     

Differential effect of isoprinosine on lymphocyte proliferation: comparison of two tissue culture media

Isoprinosine (ISP) has been reported to stimulate a variety of immunological responses. This study showed a differential effect of this drug on the proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin (PHA). ISP was found to increase this proliferative response when Eagle's Minimum Essential Medium (MEM) was used. When Medium-199 (M-199) was used to culture the cells, no increase in the proliferative response was observed. This inhibition by M-199 of the ISP effect on PHA-stimulated cells has been traced to the presence of the purines adenine and hypoxanthine in M-199.     

Corticotropin-releasing factor: effect on cerebral blood flow in physiologic and ischaemic conditions

The expression of corticotrophin-releasing factor (CRF) receptors in cerebral arteries and arterioles suggests that CRF may modulate cerebral blood flow (CBF). In the present study, the effects of CRF, CRF-like peptides and the CRF broad spectrum antagonist DPhe-CRF on CBF have been investigated under normal physiologic conditions and in the margins of focal ischaemic insult. The experiments were carried out in anaesthetised and ventilated rats. Changes in CBF after subarachnoid microapplication of CRF and related peptides were assessed with a laser-Doppler flowmetry (LDF) probe. In the ischaemic animals, agents were injected approximately 60 minutes after permanent middle cerebral artery occlusion (MCAo). Microapplication of CRF and related peptides in normal rats into the subarachnoid space produced sustained concentration-dependent increases in CBF. This effect was attenuated by co-application with DPhe-CRF, which did not alter CBF itself. A second microapplication of CRF 30 min after the first failed to produce increases in CBF in normal animals. Microapplication of CRF in the subarachnoid space overlying the ischaemic cortex effected minor increases in CBF whereas D-Phe-CRF had no significant effect on CBF. Activation of the CRF peptidergic system increases CBF in the rat. Repeated activation of CRF receptors results in tachyphylaxis of the vasodilator response. CRF vasodilator response is still present after MCAo in the ischaemic penumbra, suggesting that the CRF peptidergic system may modulate CBF in ischaemic stroke.