Adaptations to mentoring and peer mentor training at the medical faculty during the COVID-19 pandemic.

Objectives: The COVID-19 pandemic has led to major adjustments in health care systems and significantly affected medical education. Accordingly, our mentoring program MeCuM-Mentor had to expand its virtual elements, in order to continue to meet the needs for mentoring at the medical faculty of the Ludwig-Maximilians-University Munich. Methods: Here we report on our recently implemented online formats to facilitate training for currently coached peer mentors, as well as the introduction of an online consultation hour and a new social mentoring event called PubQuiz. Results: First results demonstrated feasibility of the above-mentioned virtual formats, which were positively rated by the participants in small voluntary evaluation questionnaires. Utilization rates indicate existing need for mentoring during the pandemic. In addition, the new event PubQuiz promotes social interaction among peers during isolation due to COVID-19. Conclusion: With the transition to online formats, mentoring at the Medical Faculty could be continued during COVID-19. The newly introduced mentoring event PubQuiz will be repeated. However, it remains unclear to what extent online formats can replace in-person one-to-one mentoring conversations or peer mentoring meetings.

Restructuring career counselling ventures of a mentoring program for medical students in the course of the COVID-19 pandemic.

Objectives: Due to the COVID-19 pandemic, medical curricula face major challenges. This also applies to mentoring programs, where face-to-face meetings are considered essential. Methods: The LMU Munich medical faculty mentoring program (MeCuM-Mentor) adapted to counteract the unforeseen pause of conventional course formats and associated uncertainty of many students. We here present an approach to transform the established large scale or group mentoring events of our program into online formats. Three projects are presented as examples: 1. HowTo Klink (HK), mainly informative in nature and with peer-mentoring character, 2. FacharztDuell (FAD) and 3. "Auf ein Gespräch mit... (AEGM)", both with a focus on career counseling. Results: Initial evaluations show a similarly high participation rate and a high level of satisfaction among the participating students. Students' evaluation of whether the projects presented should take place in presence or in online format has so far shown no clear trend. Conclusion: Prospective studies are necessary to investigate the effectiveness of these online formats and analyse differences in participant behaviour. The extent to which online mentoring can replace classic mentoring functions has to be discussed anew.

Disorders that mimic central nervous system infections.

Many noninfectious diseases can cause signs, symptoms, and cerebrospinal fluid (CSF) abnormalities simulating central nervous system (CNS) infection. Infection usually can be excluded in these cases by the judicious use of serologic tests and CSF stains and cultures. Then, the correct diagnosis is typically suggested by the history and the concomitant presence of clinical and laboratory evidence of disease in other organ systems. Occasionally, particularly when such evidence is absent, the distinction requires meningeal or brain biopsy.

Bispecific humanized anti-IL-2 receptor alpha beta antibodies inhibitory for both IL-2- and IL-15-mediated proliferation.

Humanized anti-Tac (HAT) and Mik beta1 (HuMik beta 1) Abs directed at IL-2R alpha and IL-2R beta, respectively, inhibit IL-2 binding and biological activity and together act synergistically in vitro. The Abs have been used successfully in primate models of allograft rejection, graft-vs-host disease, and autoimmunity. We produced bifunctional humanized anti-IL-2R alpha beta Abs (BF-IgG) to combine the specificity of the two Abs into one entity by fusing HAT-producing NSO cells and HuMik beta 1-producing Sp2/0 cells. BF-IgG was purified using protein G-Sepharose affinity chromatography, followed by IL-2R alpha and IL-2R beta affinity chromatography and hydrophobic interaction chromatography. BF-IgG exhibited both anti-IL-2R alpha and anti-IL-2R beta specificities in binding assays. While the Ab binds the IL-2R with intermediate affinity (Kd = 2.82 nM), it does not inhibit IL-15 binding to its high affinity IL-15R. In Kit225/K6 (IL-2R alpha beta gamma+) cells, BF-IgG was 10-fold more potent than a HAT/HuMik beta 1 equimolar mixture in blocking IL-2-induced proliferation and, unexpectedly, was at least 65-fold more active than the mixture in blocking IL-15-induced proliferation. This dual inhibitory activity may be due to cross-linking of the IL-2R alpha and IL-2R beta, thus blocking IL-2 binding and possibly impeding the association of IL-2R beta with IL-15R. BF-IgG has potent immunosuppressant activities against both IL-2- and IL-15-mediated responses, and this antagonist could be more efficacious than HAT and/or HuMik beta 1 for the treatment of autoimmunity and the prevention of allograft rejection.

Induction of the male-specific cytochrome P450 3A2 in female rats by phenytoin.

We previously reported that administration of dexamethasone (DEX) and other selected pharmacological agents to rats resulted in a profound increase in hepatic cytochrome P450 3A1 in both sexes, but male constitutive P450 3A2 was modestly increased (4-fold) in adult males and not detected in either treated or untreated females (Cooper et al., Arch. Biochem. Biophys. 301, 345, 1993). Using a more sensitive Western blot stain, we have now detected in females low but significant induction of P450 3A2 by DEX. Of 10 compounds tested, DEX was the most effective inducer of P450 3A1 in either sex and of P450 3A2 in males. Unexpectedly, the antiepileptic, phenytoin, was the most potent inducer of P450 3A2 in females, resulting in levels up to 30% of those seen in untreated males. Even more striking, phenytoin differentially induces the male-specific P450 3A2 with barely detectable increases in P450 3A1 in either sex. By comparison, when administered to female rats, the other active P450 3A inducers preferentially induce P450 3A1 compared to 3A2 by ratios ranging from 3- to 400-fold. Another male-specific isozyme, P450 2C11, was induced in females by both DEX and phenytoin, but DEX was much more effective than phenytoin. These results suggest that the masculinization of expression of these two sexually dimorphic isozymes of cytochrome P450 may occur by different mechanisms, and that phenytoin is atypical of the other nine compounds we tested. Moreover, of the known inducers of the "steroid inducible" 3A family, phenytoin is unique in its ability to differentially induce P450 3A2 compared to P450 3A1, particularly in the female rat. Also, administration of phenytoin to female rats gave rise to P450 3A2 levels that could be divided into two distinct classes of high and low levels of P450 3A2. Should this prove to be a genetic polymorphism, it could be very useful in studies on the mechanism of P450 3A2 induction.

Stroke due to Lyme disease.

A 56-year-old Connecticut woman suffered multiple strokes 18 months after antibiotic treatment for early Lyme disease with facial palsy. Pleocytosis, intrathecal synthesis of anti-Borrelia burgdorferi antibody, and the response to antibiotic treatment substantiated the diagnosis of neuroborreliosis. This is the first report of stroke caused by Lyme disease acquired in North America.

Monoclonal antibodies of differentiating specificities as probes of cytochrome P450h (2C11).

A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2 alpha- and 16 alpha-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine N-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetamine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.

Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats.

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P4501 (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P4501 proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16 alpha-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P4501 were detected in hepatic microsomes from immature and adult males as well as immature females but not in adult females. Cytochrome P4501 expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P4501. Hence, not only is cytochrome P4501 protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.

Human group II phospholipase A2. Characterization of monoclonal antibodies and immunochemical quantitation of the protein in synovial fluid.

Murine monoclonal and rabbit polyclonal antibodies were generated against human group II phospholipase A2 (PLA2) in order to study the role of this enzyme in inflammatory disease, the source of its synthesis, and the interaction of PLA2 with its substrate. Monoclonal antibody PLA187 exhibits potent inhibitory activity toward human PLA2 using autoclaved E. coli membranes as the substrate. Three other monoclonal antibodies (PLA184, PLA185, and PLA186) also inhibit enzyme activity, but with about 50-fold less potency. Based on the results of double-antibody competition experiments and enzyme inhibition profiles, PLA184 and PLA185 appear to recognize the same epitope. Monoclonal antibody PLA186 recognizes an epitope which is spatially distinct from that recognized by PLA184/185. The results also suggest that the epitope recognized by PLA187 may overlap with both epitopes recognized by PLA186 and PLA184/185. A double-antibody sandwich ELISA was developed using a combination of PLA185 and rabbit polyclonal antibody against PLA2. The ELISA provides a sensitive and quantitative method for monitoring specifically group II PLA2 in various biological sources, independent of factors which may affect enzyme activity. We have utilized this assay to quantitate PLA2 levels in synovial fluid from the joints of individuals with rheumatoid arthritis as well as from non-arthritic joints. Our results indicate that elevated levels of group II PLA2 in synovial fluid are not necessarily associated with arthritis.

Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D.

We have developed a simple immunoaffinity chromatography procedure for the purification of a glycosyl-phosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) from bovine serum. The enzyme was initially purified by a procedure consisting of 9% polyethylene glycol precipitation, Q Sepharose anion-exchange chromatography, S-300 gel filtration, wheat germ lectin-Sepharose, hydroxylapatite agarose, zinc chelate matrix, Mono Q-high performance liquid chromatography (HPLC), and Superose 12 (gel filtration) HPLC. Using this purified material as immunogen, we generated a panel of monoclonal antibodies. A low affinity antibody was selected for the purification of catalytically active GPI-PLD from bovine serum by immunoaffinity chromatography, followed by wheat germ lectin-Sepharose and Mono Q-fast protein liquid chromatography. The latter method provides a simple purification procedure with an overall yield of 26%. The purified enzyme has an apparent molecular weight of about 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of about 5.6 by isoelectric focusing gel analysis. On Superose 12 HPLC, the material purified by the latter method elutes as a single peak with an apparent molecular weight of 200,000 as determined by protein standards. The enzyme activity is inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid or 1,10-phenanthroline. Phosphatidic acid is the only 3H-labeled product when [3H]myristate-labeled variant surface glycoprotein is hydrolyzed by the purified enzyme. Amino terminal sequence analysis of the intact 100-kDa protein reveals no strong homology to that of any other known protein. Twelve tryptic peptides derived from the intact protein have been subjected to amino acid sequence analysis. Two of them share sequence homology with each other and with the metal ion binding domains of members of the integrin family. Based upon these criteria, it appears that the purified enzyme is distinct from other phospholipases with specificity for inositol phospholipids.

A simple, non-chromatographic purification procedure for monoclonal antibodies. Isolation of monoclonal antibodies against cytochrome P450 isozymes.

A simple, non-chromatographic purification procedure for monoclonal antibodies from mouse ascites fluid is described. This procedure, which is rapid, inexpensive, and has high capacity involves the precipitation of contaminating proteins with caprylic acid followed by precipitation of immunoglobulin using ammonium sulfate. This two-step procedure is shown to be effective for the purification of various immunoglobulins including IgG1, IgG2a and IgG2b. In the present report, more than 30 monoclonal antibodies directed against cytochrome P450 isozymes have been purified by this method and characterized.

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes.

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.

Neurologic abnormalities in Lyme disease without erythema chronicum migrans.

The clinical features in eight patients with neurologic abnormalities typical of Lyme disease and elevated titers of antibody to the spirochete, Borrelia burgdorferi, its causative agent, are described. None of the patients had the diagnostic skin lesion, erythema chronicum migrans. Lyme arthritis, the other clinical marker for the disease, developed subsequently in only three. The neurologic abnormalities included aseptic meningitis, encephalitis, cranial neuritis, motor and sensory radiculitis, and myelitis in various combinations. The occurrence of severe encephalitis resulting in dementia in two of these patients and irreversible myelopathy in one enlarges the known spectrum of neurologic abnormalities due to infection with B. burgdorferi. Lyme disease can present with neurologic abnormalities without diagnostic extraneural features, can be suspected on clinical and epidemiologic grounds, and can be diagnosed serologically.

Disorders that mimic CNS infections.

A variety of inflammatory and neoplastic disorders can cause signs, symptoms, and laboratory abnormalities suggesting CNS infection. The distinction usually can be made through careful consideration of the entire clinical picture and the judicious use of additional laboratory tests.

Hexachlorobenzene and substituted pentachlorobenzenes (X-C6Cl5) as inducers of hepatic cytochrome P-450-dependent mono-oxygenases.

Hexachlorobenzene (HCB) and 2,3,4,4',5-pentachlorobiphenyl induced a similar spectrum of cytochrome-P-450-dependent mono-oxygenase activities in the rat, including 4-dimethylaminoantipyrine N-demethylase, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD). Levels of individual cytochrome P-450 isozymes and various mono-oxygenase activities in liver microsomes from rats treated with substituted pentachlorobenzene (X-C6Cl5) and 4'-substituted-2,3,4,5-tetrachlorobiphenyl (X-C12 H5Cl4) analogues demonstrated the remarkable effects of substituent structure on induction activities. The chloro- and bromopentachlorobenzenes induced hepatic microsomal 4-dimethylaminoantipyrine N-demethylase, AHH and EROD; the iodo-, fluoro-, acetamino- and nitropentachlorobenzene analogues together with pentachlorobenzene weakly induced both AHH and EROD (approximately 2-fold or less); and the remaining substituted pentachlorobenzenes tested (X = CH3, OCH3 and OH) were relatively inactive as inducers of microsomal mono-oxygenases. Substituent effects were observed for the induction of liver microsomal cytochromes P-450a, P-450b + e, P-450c, P-450d and total cytochrome P-450 by the X-C6Cl5 and X-C12H5Cl4 analogues. The chloro- and bromopentachlorobenzene analogues in both series induced total cytochrome P-450 and cytochromes P-450a to P-450d, whereas the hydroxy-, methyl- and methoxy-substituted analogues in both series were relatively inactive as inducers of cytochrome P-450. Iodo-, fluoro- and nitropentachlorobenzene were weak 3-methylcholanthrene-type inducers and, in contrast to the corresponding biphenyl analogues, had little or no effect on the induction of cytochromes P-450a, P-450c and P-450d.(ABSTRACT TRUNCATED AT 250 WORDS)