Rehabilitation and physical activity for COVID-19 patients in the post infection period.

The first cases of COVID-19 were initially recorded in December 2019 in Wuhan, the capital of China's Hubei Province. The situation quickly escalated and turned into a global pandemic. COVID-19 is a highly infectious respiratory disease that leads to decreased respiratory, physical, and psychological function of affected patients (2). Patients' symptoms widely vary; from asymptomatic course to severe symptoms. Decrease in physical function, and, in some cases, a persistence of symptoms may be observed in patients, who overcame the infection period. Rehabilitation represents a potential treatment option for COVID-19 patients in post-infection period. Rehabilitation therapies may help to restore physical function in patients and to reduce the long-term effects of COVID19 infection (Ref. 37). Text in PDF www.elis.sk Keywords: COVID19, rehabilitation, post-infection syndrome.

Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia after first relapse.

Using flow cytometric techniques capable of detecting 0.01% leukemic cells, we prospectively studied minimal residual disease (MRD) in patients with acute lymphoblastic leukemia (ALL) after first relapse. At the end of remission reinduction, 41 patients had a bone marrow sample adequate for MRD studies; 35 of these were in morphologic remission. Of the 35 patients, 19 (54%) had MRD >/=0.01%, a finding that was associated with subsequent leukemia relapse. The 2-year cumulative incidence of second leukemia relapse was 70.2+/-12.3% for the 19 MRD-positive patients and 27.9+/-12.4% for the 16 MRD-negative patients (P=0.008). Among patients with a first relapse off therapy, 2-year second relapse rates were 49.1+/-17.8% in the 12 MRD-positive and 0% in the 11 MRD-negative patients (P=0.014); among those who received only chemotherapy after first relapse, the 2-year second relapse rates were 81.5+/-14.4% (n=12) and 25.0+/-13.1% (n=13), respectively (P=0.004). Time of first relapse and MRD were the only two significant predictors of outcome in a multivariate analysis. We conclude that MRD assays should be used to guide the selection of postremission therapy in patients with ALL in first relapse.

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.

To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-zeta-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.

Three active forms of aspartic proteinase from Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus (M-PMV) proteinase, released by the autocatalytic cleavage of Gag-Pro and Gag-Pro-Pol polypeptide precursors, catalyzes the processing of viral precursors to yield the structural proteins and enzymes of the virion. In retroviruses, usually only one proteolytically active form of proteinase exists. Here, we describe an unusual feature of M-PMV, the existence of three active forms of a retroviral proteinase with molecular masses of 17, 13, and 12 kDa as determined by mass spectroscopy. These forms arise in vitro by self-processing of a 26-kDa proteinase precursor. We have developed a process for isolation of each truncated product and demonstrate that all three forms display proteolytic activity. Amino acid analyses, as well as the determination of N- and C-terminal sequences, revealed that the N-termini of all three forms are identical, confirming that in vitro autoprocessing of the 17-kDa form occurs at the C-terminus to yield the truncated forms. The 17-kDa form and the newly described 13-kDa form of proteinase were identified in virions collected from the rhesus monkey CMMT cell line chronically infected with M-PMV, confirming that multiple forms exist in vivo.

Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.

We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.

Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli.

A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli. An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique. In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA. Selection for the antibiotic thus increases the frequency of mutants. High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector. The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed. A retroviral proteinase precursor, toxic for E. coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained.

Specificity mapping of HIV-1 protease by reduced bond inhibitors.

A series of 47 N-truncated reduced bond inhibitors, systematically modified at individual positions (P1, P'1, P'2, P'3, and P'4), were synthesized and used to map the subsite preferences of HIV-1 protease. The tight binding inhibitor of HIV-1 protease t-butoxycarbonyl-Phe-[CH2NH]Phe-Glu-Phe-NH2 (Ki = 0.2 nM) was chosen as the parent structure for further modifications. The P'2 glutamic acid was found to fit well into the S'2 subsite of the protease. The conformational restriction of any phenylalanine residue or saturation of more than one phenylalanine side chain in P'1 or P'3 lead is to a large Ki increase. Introduction of tyrosine in the P1 position improves the binding by an order of magnitude. The S'4 subsite of the protease was shown to accommodate large structural changes in the inhibitor at this position. Therefore P'4 may serve as an ideal region for further modification in order to improve bioavailability of this type of compound. An improved method of direct comparison of tight binding inhibitors with subnanomolar Ki values has been described.

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

Reduced-bond tight-binding inhibitors of HIV-1 protease. Fine tuning of the enzyme subsite specificity.

Truncation of a peptide substrate in the N-terminus and replacement of its scissile amide bond with a non-cleavable reduced bond results in a potent inhibitor of HIV-1 protease. A series of such inhibitors has been synthesized, and S2-S3' subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P1' and Glu in P2'. The S3' pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2' position yields a tight-binding inhibitor of HIV-1 protease, Boc-Phe-[CH2-NH]-Phe-Glu-Phe-OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a Ki of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1-P1' linkage for proper binding. A new type of peptide bond mimetic, N-hydroxylamine -CH2-N(OH)-, has been synthesized. Binding of hydroxylamino inhibitor of HIV-1 protease is further improved with respect to reduced-bond inhibitor.

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli.

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.