COVID-19-Associated Bifacial Weakness with Paresthesia Subtype of Guillain-Barré Syndrome.

We report a case of bifacial weakness with paresthesia, a recognized Guillain-Barré syndrome subtype characterized by rapidly progressive facial weakness and paresthesia without ataxia or other cranial neuropathies, which was temporally associated with antecedent coronavirus 2019 (COVID-19). This case highlights a potentially novel but critically important neurologic association of the COVID-19 disease process. Herein, we detail the clinicoradiologic work-up and diagnosis, clinical course, and multidisciplinary medical management of this patient with COVID-19. This case is illustrative of the increasingly recognized but potentially underreported neurologic manifestations of COVID-19, which must be considered and further investigated in this pandemic disease.

Long-term performance assessment of HPGE detectors used in the neutron activation analysis laboratory of IPEN-CNEN/SP (Brazil).

In this work, verification data for 11 HPGe detectors from two different manufacturers and three different intrinsic configurations were analyzed in respect to the stability of both the efficiency and resolution for the 122keV peak from 57Co and the 1332keV peak from 60Co. The results allow a discussion about the stability of these parameters over time (in some cases, almost 15 years), their sensitivity to imminent detector failures and their performance after a failure has been corrected; moreover, the results show a clear correlation between the manufacturer or configuration and the long-term performance of the detector.

Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.

Human epidermal cells synthesize HLA-DR alloantigens in vitro upon stimulation with gamma-interferon.

Under certain pathologic conditions, human keratinocytes synthesize and express HLA-DR antigens. Assuming that soluble mediators might be responsible for this phenomenon, differentiating, primarily DR-keratinocytes were grown in the presence or absence of mixed leukocyte culture supernatants and tested for Ia antigen expression. After 6 days of culture, keratinocytes displayed surface-bound HLA-DR alpha/beta complexes when exposed to mixed leukocyte culture supernatants but not when cultured in media alone. These HLA-DR moieties on keratinocytes result from active biosynthesis as evidenced by the demonstration of the intracytoplasmic HLA-DR gamma (invariant) chain within these cells. In view of reports that interferon-gamma promotes Ia-production in a variety of cell types, we reasoned that this lymphokine might be responsible for the Ia-inducing property of mixed lymphocyte culture supernatants. Indeed, recombinant interferon-gamma, but not interferon-alpha or interleukin-2, proved to be a potent stimulator of Ia expression by keratinocytes. The further finding that this event can be prevented by the addition of a monoclonal anti-interferon-gamma antibody strongly suggests that this cytokine is directly responsible for HLA-DR production by keratinocytes. The interferon-gamma-induced acquisition of HLA-DR antigens by primarily DR-keratinocytes may provide a useful tool to study the role of these alloantigens in T-cell activation and may also add to our understanding of mechanisms operative in epidermal cell-T-cell interactions.

Automated monoclonal radioimmunoassays for pirenzepine, a selective muscarinic receptor antagonist, in plasma and urine.

Sensitive and specific monoclonal radioimmunoassays (RIA) for pirenzepine, a muscarinic receptor (M1) antagonist, were developed and validated for rapid automated routine analysis of plasma and urine samples from clinical studies. Three discrete stable hybridoma clones secreting monoclonal antibodies (MAb) to pirenzepine were produced by fusing the myeloma line X63-Ag8.653 to spleen cells of BALB/c mice immunized with pirenzepine-5-N-propionate-protein conjugates. Of three carrier proteins investigated (HSA, BSA and edestin), optimal humoral immune responses and affinity of hybridoma antibody were attained using HSA. All three MAb displayed high affinity to pirenzepine (Ka = 0.6-1.2 X 10(9) l/mol) but showed differing cross-reactivities with its 4'-N-desmethyl metabolite (less than 1%, 6% and 40% respectively). The MAb with essentially zero metabolite cross-reactivity, 58-7/7, was selected for RIA development. In comparison, eight rabbit polyclonal antisera raised against pirenzepine-5-N-propionate-HSA or pirenzepine-5-N-butyrate-HSA possessed a similar range of affinities to the MAbs, but none approached MAb 58-7/7 in specificity. The bridge length had no significant effect on antisera characteristics. Competitive solid-phase RIAs for pirenzepine in human plasma and urine were established using MAb 58-7/7 and [3H]pirenzepine as tracer. All fluid transfers were automated using a programmable sample processing system (Microlab 2,000). Detection limits were 0.25 ng/ml (plasma) and 4 ng/ml (urine) in 0.1 ml sample. The coefficient of within-assay variation was 4% or better in the range 2-100 ng/ml (plasma) or 30-1,000 ng/ml (urine), the between-assay CV was 5.3% or better in the range 5-90 ng/ml (plasma) or 40-660 ng/ml (urine). Excellent correlation was observed between the plasma monoclonal RIA and the hitherto used polyclonal RIA (n = 106, r = 0.994), and between the urine monoclonal RIA and HPLC (n = 82, r = 0.998). We expect that these assays will prove valuable in pharmacokinetic and pharmacological investigations of pirenzepine.

Assessing consequences of marine pollution by hydrocarbons using sponges as model organisms.

Pollution has been assessed as a mutagenic activity determined by the Ames test, using radiolabelled benzo[a]pyrene (BaP) as a model pollutant. Experimental animals were sponges, mainly Tethya lyncurium from the Northern Adriatic and from the Pacific near Catalina Island, California, U.S.A. Changes in ornithine decarboxylase (ODC) activity (ODC; EC 4.1.1.17) and polyamine concentrations with and without pollution were observed. There is a slow rise in ODC activity during the course of three-weeks exposure and a fast increase of polyamine levels during the course of one day. Mixed function oxygenase (MFO; EC 1.14) activity could not be detected in sponges. There was a significant concentration dependent coupling of radioactive BaP derivatives (BaPD) to the macromolecular fractions; the highest in protein, X 1000 greater than DNA and X 500 greater than RNA. Coupling is light-mediated and drops to zero in the dark. However when activated microsomal fractions from fish, that had been exposed to high level polycyclic aromatic hydrocarbon (PAH) pollution are added, dark incorporation rises to significant levels which can be decreased by the MFO inhibitor 7,8-benzoflavone (BP). The question of possible absence of DNA repair in the sponges and some implications are discussed.

Consequences of detergent pollution of the sea: effects on regenerating sponge cubes of Geodia cydonium.

Regenerating cubes of the sponge Geodia cydonium cyconium were used as a model in the investigation of detergent pollution in the sea. The anionic detergent sodium dodecylsulphate (SDS) and a 1:1 mixture of Faks and Radion, two commercial laundry detergents, were used in the concentration range from 1 X 10(-9) g/ml (1 ppb) to 1 X 10(-5) g/ml. It is shown that SDS is taken up, weakly accumulated but not incorporated into the macromolecular fractions of the sponge. At concentrations of 0.1 ppm and above, SDS decreases the uptake of thymidine, uridine and phenylalanine into the acid-soluble sponge fraction. Their incorporation into the acid insoluble fractions, which have been isolated, was different from the controls at 10 ppb and higher levels. Faks and Radion were less active by a factor of 10. However, they showed similar effects. The chemical composition of the regenerating sponge cubes with respect to DNA, RNA and protein content has been evaluated. The alterations are less pronounced on detergent incubation than precursor uptake. The use of the cetyltrimethyl-ammoniumbromide-turbidity-dilution technique reveals drastic qualitative changes in the nucleic acid fractions. The relevant literature on biological effects of detergent is listed. It is shown that this investigation extends the scale of known effects far into the low and pollution-relevant concentration levels.

Determination of neutron flux distribution in an Am-Be irradiator using the MCNP.

A neutron irradiator has been assembled at IPEN facilities to perform qualitative-quantitative analysis of many materials using thermal and fast neutrons outside the nuclear reactor premises. To establish the prototype specifications, the neutron flux distribution and the absorbed dose rates were calculated using the MCNP computer code. These theoretical predictions then allow one to discuss the optimum irradiator design and its performance.

Measurements of k0 and Q0 values for 64Zn(n,γ)65Zn and 68Zn(n,γ)69mZn reactions with covariance analysis.

The values of k(0) and Q(0) for (64)Zn(n,γ)(65)Zn and (68)Zn(n,γ)(69m)Zn reactions were determined experimentally. The irradiations were performed near the core of the IEA-R1 3.5MW nuclear research reactor of the Nuclear and Energy Research Institute - IPEN-CNEN/SP, in São Paulo, Brazil. The results for the neutron field parameters f and α were 49.7(19) and -1.1(31)×10(-3), respectively. The resulting values of k(0) and Q(0) for (64)Zn(n,γ)(65)Zn reaction were 5.63(8)×10(-3) and 1.69(6), respectively, and the corresponding values for (68)Zn(n,γ)(69m)Zn reaction were 4.00(6)×10(-4) and 2.34(4), respectively. These results were compared with the literature.

Pharmacokinetics of tumor necrosis factor alpha after intravenous administration in rats. Dose dependence and influence of tumor necrosis factor beta.

2, 10, 20, 100 and 500 micrograms/kg/0.5 h of recombinant human tumor necrosis factor alpha (TNF-alpha) were administered to rats by intravenous infusion over 30 min. At high doses (greater than 10 micrograms/kg/0.5 h) the plasma half-life (t1/2, about 0.5 h) of TNF-alpha was practically dose independent. In contrast at the lowest dose (2 micrograms/kg/0.5 h) the elimination of TNF-alpha was markedly faster: A plasma half-life of only 5-8 min was determined. However, when in addition to 2 micrograms/kg/0.5 h of TNF-alpha a high dose of recombinant human tumor necrosis factor beta (TNF-beta) was infused the plasma half-life was enhanced up to values at high doses (greater than 10 micrograms/kg/0.5 h). Thus at the lowest dose the speed of elimination of TNF-alpha was enhanced which could be prevented by TNF-beta.

Control of the initiation of DNA replication in Escherichia coli. II. Function of the dnaA product.

General growth parameters and the kinetics of DNA replication have been determined in merogenotes carrying different combinations of the dnaA+ and the dnaA5 allele. The strain which is homozygous diploid for dnaA5 is different from all other combinations in cell volume, DNA per mass ratio, number of replication points per chromosome, and polymerization rate of DNA. From this we deduce that the dnaA product is a positively acting regulatory protein in initiation. In an appendix we show that in combinations between the dnaA5 and dnaA204 alleles the phenotype of dnaA5 is dominant.

Pharmacokinetics of recombinant human tumor necrosis factor alpha in rhesus monkeys after intravenous administration.

Recombinant human tumor necrosis factor alpha (TNF-alpha) was administered to rhesus monkeys by i.v. short-term infusions (0.5 hr) of 10, 20, 30 and 120 micrograms/kg and long-term infusions (6.5 hr) of 22, 54, 135 and 325 micrograms/kg. At high plasma levels of TNF-alpha (doses of 120 micrograms/kg of short-term infusions and greater than or equal to 54 micrograms/kg of long-term infusion) the pharmacokinetics of TNF-alpha were practically first order. A plasma T1/2 of 1.2 to 2.1 hr was calculated. However, at low plasma levels (doses of 10-30 micrograms/kg of short-term infusion and 22 micrograms/kg of long-term infusion) the elimination rate increased steadily in dependence on concentration and time. We conclude that at low concentrations of TNF-alpha the pharmacokinetics were not first order. Simultaneous long-term infusion of recombinant human TNF-beta (200 micrograms/kg) in addition to 22 micrograms/kg of TNF-alpha reduces the elimination rate of TNF-alpha, which can be concluded from the elevation of the TNF-alpha plasma levels. Furthermore, there was no time-dependent increase of the elimination rate that was detected without infusion of TNF-beta. Based on these results two different elimination mechanisms of TNF-alpha in rhesus monkeys are postulated: an unspecific, nonsaturable process as well as a specific, saturable mechanism.

Investigation of a tetracycline-regulated phage display system.

A new vector (pGZ1) was developed for bacterial phage display of antibody fragments using a transcriptional regulation element with tight control. The tet(p/) degrees -based phasmid exhibits fully suppressed scFv background synthesis in the absence of inducer and is independent of glucose as a catabolite repressor. The vector is shown to be a useful alternative to commonly used lac(p/) degrees -regulated systems.

Control of the initiation of DNA replication in Escherichia coli. I. Negative control of initiation.

A transient stimulation of the initiation of DNA replication during inhibition of protein synthesis has been demonstrated in dnaA5 and dnaA46 mutants. This suggests the existence of a negatively acting control protein (not the dnaA product) which decays rapidly or is removed during a block in protein synthesis. The stimulation of initiation is dependent on a previous accumulation of initiation specific proteins, which suggests that in addition to the negative control other control mechanisms are effective.

Pharmacokinetics and biological activity in subcutaneous long-term administration of recombinant interferon-gamma in cancer patients.

We have investigated the pharmacokinetics, tolerance, and biological activity of recombinant human interferon-gamma (rHuIFN gamma) administered subcutaneously to cancer patients. Twenty-one patients with lymphoma and metastatic cancer received rHuIFN gamma (in doses of 0.1, 0.25, or 0.5 mg/m2) in two or three injections per week for up to 180 days. The most common adverse effects encountered were flu-like symptoms, fever and fatigue. The increase in body temperature after each administration ranged from 0 to 4 degrees C depending on the individual patient, but was unrelated to the rHuIFN gamma dose or its plasma concentration. The pharmacokinetic response of the patients after the two treatments showed a low intra-individual variability with respect to the plasma concentration/time profiles. However, as observed for the fever side-effect, the interindividual variation (CV greater than 50%) was high for the parameters area under the data points (AUC0-t) and maximum plasma concentration (cmax). Despite this high interindividual variability, the mean values obtained for AUC0-t and cmax after s.c. injection of rHuIFN gamma were approximately proportional to the dose administered: the injection of 0.1, 0.25 or 0.5 mg/m2 rHuIFN gamma resulted in AUC0-t values of 15.4, 31.5 or 69.6 ng h/ml, respectively and cmax was found to be 1.0, 2.4 and 4.9 ng/ml, respectively. With this s.c. administration protocol, objective antitumour responses were observed in two patients, but there was no partial or complete remission.

Trans-dominance of dnaA mutants in Escherichia coli.

Temperature sensitivity of growth and DNA synthesis was tested in merogenotes heterozygous for the dnaA allele. All combinations tested (F dnaA+/dnaA5, F dnaA+/dnaA46, F dnaA+/dnaA204, F dnaA5/dnaA+, F dnaA204/dnaA+) were temperature sensitive. The mutant dnaA allele is thus trans-dominant to the wild type allele.

Expression of Qat-4 and Qat-5 alloantigens on cytotoxic precursor and effector cells: different surface phenotypes of alloreactive and H-2 restricted cytotoxic T cells.

Monoclonal anti-Qat-4 and anti-Qat-5 antibodies, which define antigens expressed on peripheral T cell subsets, have been used to study the phenotypes of alloreactive and H-2-restricted cytotoxic effector cells and their precursors. Depletion of Qat-4+ or Qat-5% cells from the T cell pool prior to their sensitization in bulk cultures prevented the development of alloreactive and H-2-restricted cytotoxic activities in the selected populations. No reconstitution of cytolytic activities to normal levels was obtained when mixtures of Qat-4- and Qat-5- cells were sensitized in bulk cultures to H-2 or non-H-2 antigens. Sensitization of limiting numbers of Qat-4- or Qat-5- lymphocytes under optimal conditions for help (interleukin 2), with the appropriated antigens (H-2 or H-Y) did not result in the generation of cytotoxic T cells, indicating that the majority of all cytotoxic T lymphocyte (CTL) precursors are Qat-4+, Qat-5+. When CTL effector populations were treated with the antisera and complement (C) at their maximum CTL activity, it was found that H-2-restricted CTL were totally eliminated by anti-Qat-4 and considerably reduced by anti-Qat-5 antisera and C. In contrast, alloreactive CTL effector cells were insensitive to anti-Qat-4 and to anti-Qat-5 plus C. Although alloreactive CTL effector populations regained some Qat-4 antigens during further in vitro culture, it was shown that H-2-restricted CTL were at all times more sensitive to anti-Qat-4 than were alloreactive CTL. The findings suggest that during maturation of alloreactive and H-2-restricted CTL from their precursors, both alloantigens undergo differential quantitative variations in their expression that lead to different Qat-4,5 phenotypes of alloreactive and H-2-restricted CTL.

Immunoenzymatic assessment of interferon-gamma in Hodgkin and Sternberg-Reed cells.

The typical histological picture seen in Hodgkin's disease is consistent with the release of cytokines and other active mediators by the malignant cells, i.e., Hodgkin and Sternberg-Reed cells. Since interferon-gamma is regarded as an important regulator of the cytokine cascade, we have undertaken an immunohistological assessment of this mediator in Hodgkin's disease tissue biopsies. In approximately 50% of the cases investigated we found Hodgkin and Sternberg-Reed cells to be positive with antibodies against interferon-gamma. These in situ findings were substantiated by immunostaining of Hodgkin's disease-derived cell lines L428 and L540. L540 was consistently positive, whereas L428 was negative. It is noteworthy that L428 exhibit a B-cell pheno- and genotype, whereas L540 is of T-cell origin. These data are consistent with theories that propose that cytokine production by tumour cells is central to the pathogenesis of Hodgkin's lymphoma.