Pathogenesis of pelvic pain syndrome associated with endometriosis in patients resistant to surgical treatment.

AIM: Endometriosis is one of the most common gynecological diseases diagnosed in almost 70% of patients with chronic pelvic pain (CPP). However, a quarter of women with pelvic pain is diagnosed with external genital endometriosis (EGE) during laparoscopy. A special group is represented by patients with PP that did not stop after the removal of endometrial foci. The mechanisms of the pathogenesis of the formation of pain syndrome are not completely explored yet. According to several authors, a significant role in the pathogenesis of pelvic pain recurrence after surgical treatment of EGE is played by active neuroangiogenesis, both in ectopic and eutopic endometrium. The aim of the study was to expand the understanding of the pathogenesis of pelvic pain that did not stop (recurrence) after surgical treatment of external genital endometriosis. MATERIAL AND METHODS: The study involved 2 stages. At the first stage (algological), data from B&B, NRS and VRS algological questionnaires, which were completed by patients with recurrent PP after surgical treatment of EGE, were analyzed (n = 130, aged 18-45 years old, average age 32.5 ± 7.6 years). All women were operated on for EGE no later than 3-6 months after assessing the patients by the algological questionnaires; they did not receive drug therapy after surgical treatment and sought medical attention for recurrent pelvic pain. Materials for the study of the endometrium were obtained by the pipelle biopsymethod. The control group was formed from a number of women with EGE without PP, who applied for surgical treatment of infertility (n = 30). RESULTS: The results of the study have shown that the basis of pathogenesis of pelvic pain recurrence in patients who did not receive medical therapy after surgical treatment of EGE is the activation of neuro-angiogenesis processes and reduction of apoptosis. The results show a statistically significant 1.6 times increasing expression of NGF in eutopic endometrium (57.9 ± 2.5 vs 35.3 ± 2.1% of patients with the silent form of the gene and its receptor NTRK1 1.8 times (2.78 ± 0.25 versus 1.56 ± 0.21.e. respectively). Conclusion: The pathogenesis of pelvic pain in patients who did not receive medical therapy after surgical treatment of endometriosis compared to no pain form of the disease is the activation of the processes of neurogenesis in the eutopic endometrium.

Oxytocinergic regulation in pathogenesis of pelvic pain caused by adenomyosis.

OBJECTIVE: The aim of the study was to expand the understanding of pathogenesis of adenomyosis-associated pelvic pain. MATERIAL AND METHODS: We studied 30 (n = 30) biopsy samples obtained after hysterectomy in women with diffuse adenomyosis of grade II-III, accompanied by severe pain syndrome, who did not receive hormonal therapy. The morphologic comparison group comprised 30 (n = 30) biopsy samples obtained from women with adenomyosis, without pain syndrome, operated on for abnormal uterine bleeding, who also did not receive hormone therapy. RESULTS: The total density of immunological OTR labeling in the adenomyotic lesion foci was 73.7 ± 1.8%, and in the morphological control group it was 35.2 ± 1.4% (p <0.05), which indicates a significant effect of oxytocin as a ureterotonic peptide. Processes of local neurogenesis and growth of nerve fibers was established due to an increase in the expression of the nervous system growth factor NGF in the myometrium stroma, in comparison with biopsy samples of morphological control.Conclusion: Pelvic pain pathogenesis in women with diffuse adenomyosis compared with the painless form of the disease is an increase in the activity of ureterotonic factors of OTR oxytocin. Compared to the painless form of adenomyosis, the myometrial innervation apparatus of patients with pelvic pain is characterized by a significantly higher expression of nerve growth factor.

Versatile format of minichaperone-based protein fusion system.

Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus' E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification.

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.

High-efficient expression, refolding and purification of functional recombinant C-terminal fragment of human alpha-fetoprotein.

Human alpha-fetoprotein (hAFP) is an oncofetal protein which is a common cancer marker. Conjugates of native hAFP with different cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The hAFP interacts with its receptor (AFPR) on the surface of cancer cells via its C-terminal domain. The aim of this work was to develop a highly efficient expression system in Escherichia coli and efficient refolding procedure for the recombinant C-terminal fragment of hAFP (rAFP-Cterm) and to characterize its functional properties. C-terminal fragment of hAFP (rAFP-Cterm) comprising amino acids from 404 to 609 was expressed in E. coli BL21 (DE3) strain with high yield. High efficient purification and refolding procedures were developed giving yield of refolded protein about 80% with purity about 95%. The refolded rAFP-Cterm bound specifically with cancer cells carrying AFPR and was accumulated by them with the same efficiency as native hAFP. This rAFP-Cterm can be used as a vehicle for the targeted delivery of drugs to cancer cells.