Enzymatic synthesis of penicillins

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.     

Phenotypic and functional characteristics of tumor-associated lymphocytes in patients with malignant ascites receiving intraperitoneal infusions of recombinant interleukin-2 (rIL-2)

In the course of a phase I trial, in which recombinant IL-2 (rIL-2) was infused intraperitoneally (i.p.) in patients with peritoneal carcinomatosis, we evaluated the effect on "tumor-associated lymphocytes" (TAL) isolated from the ascitic fluid. No major changes in the percentages of cells expressing the CD3, CD4, CD8, Leu-7, OKM1 and WT-31 antigens were detected either in TAL or in peripheral blood lymphocytes (PBL) after 7 days of rIL-2 infusion. In contrast the percentages of TAL (but not PBL) expressing surface IL-2 receptor (Tac), or LAK-1 antigen were sharply increased. Analysis of cytolytic functions showed a potentiation of the lytic activity against natural-killer (NK) sensitive K562 target cells and the de novo appearance of lytic activity against fresh melanoma cells. In one patient IFN-gamma was detected in the ascitic fluid following rIL-2 infusion. T-cell clones derived from the patient were analyzed for the IFN-gamma production. While only approximately 40% of PB-derived control clones produced medium to low amounts of IFN-gamma, all of the TAL-derived clones produced medium to high amounts of the lymphokine.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

Effects of enzyme replacement therapy on pain and health related quality of life in patients with Fabry disease: data from FOS (Fabry Outcome Survey)

Background: Fabry disease is an X linked lysosomal storage disease caused by deficiency of the lysosomal enzyme α-galactosidase A. This leads to accumulation of globotriaosylceramide in nearly all tissues, including the blood vessels, kidney, myocardium, and nervous system. Symptoms often begin in childhood and include acroparaesthesia, with burning or tingling pain that spreads from the extremities to more proximal sites.

Aims: This study set out to evaluate pain and its influence on quality of life in patients with Fabry disease receiving enzyme replacement therapy (ERT) with agalsidase alfa.

Methods: Data were obtained from the Fabry Outcome Survey. Pain was measured using the Brief Pain Inventory (BPI), and health-related quality of life (HRQoL) was documented with the European Quality of Life Questionnaire (EQ-5D).

Results: The mean (SD) score for "pain at its worst" on the BPI prior to ERT was 5.1 (2.7). One year after commencement of ERT, this had improved by 0.5, and improved by a further 0.6 after 2 years (p<0.05). Similar statistically significant improvements were seen for "pain on average" and "pain now" after 2 years of ERT. The mean HRQoL utility score prior to ERT was 0.66 (0.32). After 12 months of treatment with agalsidase alfa, this had improved to 0.74 (0.26; p<0.05); this improvement was maintained after 2 years.

Conclusions: ERT with agalsidase alfa significantly reduces pain and improves quality of life in patients with Fabry disease.

     

Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (Mh) is the primary agent of porcine enzootic pneumonia (PEN), a chronic respiratory disease endemic to pig farms, and characterized histologically by infiltration of mononuclear cells in airways and prominent hyperplasia of the bronchus-associated lymphoid tissue (BALT). To gain further insight into the pathogenesis of PEN, cytokine expression in the lung, with particular attention to the BALT, was examined immunohistochemically in pigs naturally infected with Mh. An increase (P < 0.05) in proinflammatory and immunoregulatory cytokines (especially interleukin [IL]-2, IL-4 and tumour necrosis factor [TNF]-alpha, and to a lesser extent IL-1 [alpha and beta] and IL-6) was detected in the BALT, which showed intense lymphoid hyperplasia. IL-1beta and TNF-alpha were also detected in the bronchoalveolar exudate of infected pigs, and IL-6 and IL-8 were demonstrated in mononuclear cells of the alveolar septa. The results showed that in Mh infection, macrophage and lymphocyte activation results in the expression of a number of cytokines capable of inducing lung lesions and lymphoreticular hyperplasia of the BALT.     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

HLA antigens in ulcerative colitis: a study in the Sicilian population

HLA antigens were investigated in 41 Sicilian patients with ulcerative colitis and in 151 healthy controls. Frequencies of HLA-B5 and DR2 were increased in the group of patients with ulcerative colitis whereas the DR3 antigen frequency was decreased. However the corrected p values were not significant. Thus, present results indicate that in ulcerative colitis HLA linked genetic factors play a marginal role, if any.