MRP2 (ABCC2, cMOAT) expression in nuclear envelope of primary fallopian tube cancer cells is a new unfavorable prognostic factor.

OBJECTIVE: To determine the prognostic value of the immunohistochemical evaluation of the multidrug resistance-associated protein 2 (MRP2) expression, together with its subcellular localization in primary fallopian tube carcinomas (PFTCs). METHODS: The immunohistochemical analysis was performed using samples originating from 70 patients with PFTCs. RESULTS: (1) We documented that MRP2 can be localized in the plasma membrane (MRP2c), as well as in the nuclear envelope (MRP2n) of the PFTC cells. (2) Patients with more advanced stage, with progression of the disease and patients who died, showed significantly higher expression of the MRP2n. (3) Univariate and multivariate analyses showed that MRP2n is an unfavorable prognostic factor in PFTCs. (4) The analysis of the classic clinicopathological data revealed that only the FIGO stage had prognostic value, both in the univariate, as well as in multivariate analysis. CONCLUSIONS: (1) This study suggests that MRP2n is a new disadvantageous prognostic factor in PFTCs and (2) that expression in nuclear envelope can be associated with lower differentiation of cancer cells and their resistance to the cisplatin. (3) We have also confirmed independent prognostic value of FIGO stage in PFTCs.

The effect of smoking on expression of IL-6 and antioxidants in pancreatic fluids and tissues in patients with chronic pancreatitis.

OBJECTIVES: The present study was conducted to ascertain whether cigarette smoke induces oxidative stress in the human pancreas concurrently with inflammation. MATERIALS AND METHODS: The influence of tobacco smoking on the serum level of interleukin-6 (IL-6), on the activities of glutathione peroxidase (GPx) and copper-zinc superoxide dismutase (Cu/Zn SOD) as well as on the metallothionein (MT) level in the pancreatic pseudocyst fluid and its immunohistochemical localization in tissues of non-smoking (n = 9) and smoking (n = 12) patients with diagnosed chronic pancreatitis (CP) was measured. The concentration of interleukin-6 and metallothionein was determined by means of ELISA and the radioisotopic method, respectively. The enzyme activities in the fluid were assayed by the colorimetric method. Samples of tissues of normal pancreas (n = 4) and CP (non-smoking n = 7; smoking n = 12) were verified histopathologically and then IL-6, MT and enzymes were localized by immunohistochemical staining using the monoclonal anti-human antibody. RESULTS: The concentrations of metallothionein and interleukin-6 were significantly higher in smoking patients with CP (as compared with the non-smoking population (p < 0.01; p < 0.001). Interestingly, the ratio of MT/IL-6 in smoking patients with CP was reduced in comparison to non-smoking patients (respectively: 1.1; 5.6). In smoking patients, a significant elevation of the Cu/Zn SOD and GPx activities was revealed as compared with the non-smokers (p < 0.04; p < 0.0007). These studies clearly demonstrate a moderate and strong expression of IL-6 and enzymes in acinar, islet and duct cells of smoking patients. CONCLUSIONS: These observations favor the role of the oxidative stress in the induction of pancreatitis associated with chronic cigarette smoke inhalation.

Expression of MMP-2, TIMP-2, TGF-ß1, and decorin in Dupuytren's contracture.

To investigate the mechanisms underlying matrix deposition in Dupuytren's disease, the expression of gelatinase A (MMP-2), the tissue inhibitor of metalloproteinase-2 (TIMP-2), transforming growth factor beta 1 (TGF-ß1), decorin (DCN), and periostin was studied. The level of relative MMP-2 activation was investigated using zymography. The mRNA expression of MMP-2, TIMP-2, TGF-ß1, and DCN was detected using reverse transcription polymerase chain reaction (RT-PCR), while the presence of protein was detected using immunohistochemical (IHC) and Western blot techniques. The level of MMP-2 activation was significantly elevated in tissues with Dupuytren's contracture. RT-PCR demonstrated significantly higher expression of MMP-2, TIMP-2, TGF-ß1, and DCN mRNA in the pathological tissues; and the IHC and immunoblotting studies revealed elevated expression of TGF-ß1, DCN, and periostin. The balance between MMP-2 and TIMP-2 was disrupted in patients with Dupuytren's disease. TGF-ß1, DCN, and periostin are involved in extracellular matrix (ECM) homeostasis in Dupuytren's contracture.

Immunohistochemical localization of interleukin-6 in human pancreatitis.

The aim of the study was to identify immunohistochemically the localization of interleukin (IL)-6 in normal pancreas and in chronic pancreatitis (CP). Samples of tissues of normal pancreas (n=5) and CP (n=16), were verified histopathologically and then IL-6 was localized by immunohistochemical staining using the monoclonal antihuman IL-6 antibody and test LSAB2-HRP to visualize IL-6/Ab complexes. In slices of the pancreas, derived from patients with CP, a much stronger immunohistochemical reaction was noticed as compared with controls specimens. IL-6 was localized in exocrine, islet cells and ducts cells of the pancreas. Interestingly, this cytokine was detected in cytoplasm and very close to nucleus. Moreover, in cases of CP with inflammatory infiltration, there were a markedly stronger IL-6 expression, than that observed in specimens without infiltrate. In conclusion, the results presented herein clearly demonstrated a moderate and strong expression of IL-6 in exocrine and endocrine cells of patients with CP. These observations provide further support for the existence of local immune-pancreatic interactions.

Immunohistochemical study of decorin expression in polyps and carcinomas of the colon.

BACKGROUND: Recent studies showed that a small leucine-rich proteoglycan, decorin, may suppress tumor progression as a natural anticancer agent negatively controlling cellular growth. It was hypothesized that physiological expression of decorin may be associated with cellular senescence of the colorectal mucosa and that its down-regulation, promoting an increase in cellular proliferation, could participate in the progression of adenoma to adenocarcinoma. Therefore the expression of decorin in hyperplastic and neoplastic polyps of the colorectum was examined and compared with normal colonic mucosa and colon cancer tissues. MATERIAL/METHODS: Tissue samples were obtained from 41 patients with different types of colonic polyps (6 hyperplastic adenomas, 34 neoplastic adenomas, and 1 adenomatous polyp with focal carcinoma) and 12 patients with colon cancer. Seven samples of normal colon tissue were used as controls. Paraffin-embedded samples were used for immunohistochemical study. RESULTS: Normal and hyperplastic tissues and the majority of tubular adenomas showed strong expression of decorin in the stroma. Adenomas with a villous component showed moderate and very low decorin immunoreactivity. The decrease in decorin reactivity in tubulo-villous adenomas was significant as compared with other polyps and controls. Weak decorin immunoreactivity in stroma adjacent to clusters of cancerous cells was also found in most cases of common types of adenocarcinoma, but not in adenocarcinoma mucinosum. CONCLUSIONS: The expression of decorin may be involved in the differentiation of colonic polyps and reduced expression of decorin may abrogate the defensive potential of stromal tissue and promote the development of common types of colon carcinoma.

Cytokine gene expression in kidney allograft biopsies after donor brain death and ischemia-reperfusion injury using in situ reverse-transcription polymerase chain reaction analysis.

BACKGROUND: This study focuses on the cytokine genes expression after brain-death, ischemia-reperfusion injury, and during allograft rejection. METHODS: A total of 49 needle core biopsies from kidney transplant recipients, performed before and during transplantation procedures were studied. The first biopsy was taken during procurement of the organ, the second after cold ischemia, and the third after approximately 30 min of reperfusion. We also assessed 34 allograft biopsies obtained during acute rejection. Tubular and glomerular expression of interferon (IFN)-gamma, transforming growth factor (TGF)-beta1, platelet-desired growth factor-B (PDGF-B), interleukin (IL)-2, IL-6, IL-10 mRNA was analyzed with reverse-transcription polymerase chain reaction (RT-PCR) in situ technique, which allows to detect a few copies of the target gene without destruction of the tissue architecture. RESULTS: Compared with normal kidney tissue from living donor, high gene expression of IFN-gamma, TGF-beta1, PDGF-B, IL-2, IL-6, and IL-10 was detected in all procurement specimens. After reperfusion gene expressions of IL-2, IL-6, and IL-10 were significantly upregulated in renal tubules compared to biopsies taken after cold ischemia. The gene expression of IFN-gamma, TGF-beta1, and PDGF-B remained stable after organ procurement, during cold ischemia, and after reperfusion. Gene expression of IFN-gamma, IL-2, IL-6, IL-10, and PDGF-B in procurement biopsies, as well as in those taken after cold ischemia and reperfusion, were significantly higher than during the period of acute rejection. CONCLUSION: The data presented herein strongly point out the importance of the immunological and morphological injury that occurs before and during transplantation. The increase of inflammatory response after brain death is important for further stimulation of the immune response and long-term kidney survival.

Radiation-induced brachial plexus neuropathy - aetiopathogenesis, risk factors, differential diagnostics, symptoms and treatment.

The success of radiation oncology has led to longer patient survival. This provides a greater opportunity for radiation injuries of the peripheral nerves to develop. Brachial plexus neuropathy in cancer patients may result from either tumour recurrence or as a consequence of radiation therapy. Distinguishing between radiation injury and cancer disease recurrence as a cause of brachial plexus dysfunction may be difficult. In this article the most important principles of the differential diagnostics have been presented. Furthermore the aetiopathogenesis of brachial plexus neuropathy after radiotherapy has been discussed as well as main risk factors, symptoms of plexopathy and methods of treatment. It ought to be emphasized that complications of radiation therapy sometimes occur many years after treatment and this may create difficulties in initial diagnostics.

Brachial plexus injuries after radiotherapy - analysis of 6 cases.

Radiation-induced brachial plexus neuropathy is caused by compression of the nerve fibres by dense and inelastic fibrous connective tissue. In this study our own experience in treatment of lesions of the brachial plexus after radiotherapy is presented. The clinical material consisted of 6 patients aged from 40 to 64 years with injuries of the brachial plexus after radiotherapy. The analysis of the material comprised: basic disease, duration of radiotherapy, radiated fields, total dose of radiation, onset and character of symptoms, location and severity of injury. 5 women were qualified for surgical treatment. After neurolysis of the brachial plexus a significant improvement was obtained in 2 cases. In one patient remission of pain and sensory recovery was temporary. No improvement was observed in the remaining 2 patients. Lesions of the brachial plexus after radiotherapy are rare but difficult to prevent. The treatment depends on the grade of severity of injury. Surgical neurolysis is advised for grades 3 and 4 on the LENT-SOMA scale.

[Intraneural ganglion of the tibial nerve--a case report]. / Torbiel galaretowata nerwu piszczelowego--opis przypadku.

This paper describes a surgically treated case of gelatinous cyst of extremely rare location within the tibial nerve at the level of the popliteal fossa. The initial diagnosis was based on imaging exams (MRI, ultrasonography) and surgical exploration of the tumour. The intraneural ganglion was completely removed. Neurological motor and sensory deficits were observed neither in the preoperative nor in the postoperative period. The ultimate diagnosis was established based on the histopathological examination.

[The evaluation of the properties of the silicone-rod and tissue reation after 5,5 years period of implantation]. / Ocena wlasciwosci preta silikonowego oraz odczynu tkanek po 5,5 letnim okresie implantacji.

In this study we presented a case of prolonged detention of the temporary flexor tendon prosthesis after implantation in the hand. The silicone-rod removed after more than 5 years was subject to an examination: scanning microscopy, measurement of hardness, scanning differential calorimetry, spectroscopy in infra-red and resistance examinations. The obtained results were compared with a findings after examinations of the new, not used silicone-rod. The greatest changes were observed in maximum value of tensile strength (sigmaB) of the material after test of uniaxial tensile tests, which was about 30% smaller for a silicone-rod after implantation. The other result of investigations didn't reveal an important differences between a new and a used rod. The comparison of the tissue reaction was performed by collection a part of sheath in described case and a part of sheath produced around a rod after 10 weeks period of implantation. The generation of a capsules consisted of fibrous connective tissue with concomitant inflammation process was observed in both cases in histopathological view. Silicone rubber is a material which preserve its most important properties even after prolonged period of implantation.

[The role of matrix metalloproteinases in tumor invasion and metastasis]. / Rola metaloproteinaz macierzowych w procesie inwazji nowotworowej i przerzutowaniu.

The complicated process of invasion and metastasis consists of a long series of sequential and interrelated steps. The outcome of the process is dependent on both: the tumour cells and the properties of tissue microenvironments. Many investigators are interested in the influence of extracellular matrix components on that process. Especially matrix metalloproteinases (MMPs)--family of zinc-dependent enzymes, which take part in the coordination of extracellular matrix synthesis and breakdown seems to play crucial role in this process. A positive correlation between different type of MMPs and specific tumors has been demonstrated in many studies. In this article we summarize the current views on the role of MMPs in cancer invasion and metastasis.

[Gene expression of INF-gamma, IL-10, IL-2, IL-6, PDGF-B i TGF-beta in kidney tissue after renal transplantation]. / Ekspresja genów dia: IFN-gamma, IL-10, IL-2, IL-6, PDGF-B i TGF-beta w wycinkach nerek od biorców przeszczepu nerki.

UNLABELLED: The aim of the study was to evaluate the effect of brain death, ischemia-reperfusion injury and alloreactivity of some cytokines on intragraft mRNA expression. MATERIAL AND METHODS: We have examined 49 needle core biopsies of kidney allografts from cadaveric donors. Samples were taken before harvesting, after cold ischemia and approximately after 20-30 minutes of reperfusion. We have also assessed 56 allograft biopsies taken after transplantation. Tubular and glomerular expression of IL-2, IL-6, IL-10, IFN-gamma, TGF-beta 1 and PDGF-B mRNA was assessed using semiquantitative evaluation of the RT-PCR in situ on paraffin tissue sections. RESULTS: In all pre-procurement specimens high glomerular and tubular IL-2, IL-6, IL-10, TGF-beta 1, PDGF-B and IFN-gamma mRNA expression was detected. After reperfusion an increase of IL-2, IL-6, and IL-10 mRNA expression was observed in all specimens and was limited only to tubules. Biopsies samples with borderline changes exhibited the lowest levels of cytokine gene expression close to the intensity in control specimens. An intense, comparing to normal kidney, tubular and glomerular all examined cytokines gene expression was also noticed during acute rejection. No significant differences between acute cellular and vascular rejection were noticed. The mRNA expression of IFN-gamma and IL-2, IL-6, IL-10 in chronic rejection did not differ from acute rejection. The tubular expressions of mRNA for IL-6 and TGF-gamma 1 in biopsies with acute rejection obtained from patients treated with MMF were significantly lower than in biopsies obtained from patients treated with azathioprine.

[Immunohistochemical localization of p53 protein in smoking patients with chronic pancreatitis]. / Immunohistochemiczna lokalizacja bialka p53 u palacych pacjentów z przewleklym zapaleniem trzustki.

Epidemiological studies show that smokers are at a significantly higher (70%) risk of getting affected by chronic pancreatitis and tumours of pancreas than non-smokers. The aim of the study was to assess the effect of cigarette smoking on the expression of gene p53 in pancreas by defining immunohistochemical localization of p53 protein in tissue specimens of the pancreases derived from healthy persons and smoking and non-smoking patients with diagnosed chronic pancreatitis (CP). Patients underwent surgery in which tissue material was collected. Immunohistochemical localization of 53 protein in paraffin tissue specimens of the pancreas was performed using the LSAB2-HRP visual test (DAKO K0673) with monoclonal p53 protein (DAKO N1581) antibodies. Exposure to tobacco smoke was assessed by determining cotinine in the patients' serum using the ELISA method. The study revealed a expression of p53 protein in smoking CP patients. In non-smoking patients and healthy persons wasn't showed expression of protein. Smoking patients above the twenty cigarettes for day showed significantly higher expression of p53 protein in the pancreas compared to smoking patients less. Cigarette smoking increases the expression p53 protein in pancreas of smoking patients with chronic pancreatitis. Impairment of the gene p53 in pancreas is frequently manifested by complications in pancreatitis resulting among others from long-term smoking, and conducted by tumour proliferation.

The analysis of the intra-surgical view of the obstetric brachial plexus palsy.

The surgical intervention is necessary in about 20% of all cases of the perinatal brachial plexus palsy. In this study the intra-surgical view and the applied microsurgical techniques were analysed. The clinical material consisted of 49 children with the obstetric brachial plexus palsy treated operatively, on which the following changes were found intra-surgically: compression of the brachial plexus in 21 cases and injuries with discontinuity of the elements of the brachial plexus in 28 cases. The following surgical procedures were performed: neurolysis - 31 cases, direct neurorrhaphy - 7 cases, 2 - 4 sural nerve grafting from 2 to 3 cm - 4 cases, extra-anatomical extra-plexual reconstruction - 6 cases, extra-anatomical intra-plexual reconstruction - 1 case. In all cases the intra-surgical view motivated the operative intervention. The most important problem in treatment of the obstetric brachial plexus palsy is a separate group of children who need a surgical intervention.

The lower extremity nerve injuries - own experience in surgical treatment.

The frequency of the lower extremity nerve injuries is assessed to about 20% of the overall lesions to the peripheral nerve system. Peroneal neuropathy is the most common lower extremity nerve palsy. In this study, results of the surgical treatment of the lower extremity nerve injuries have been presented. The clinical material consisted of 270 patients (192 males, 78 females aged from 3 months to 74 years) with injuries of the common peroneal nerve - 125, sciatic nerve - 93, common peroneal and tibial nerve - 21, tibial nerve - 17, femoral nerve - 10, others - 4. The following surgical procedures were performed: external neurolysis - 164, internal neurolysis - 27, reconstruction with sural nerve grafting - 63, direct neurorrhaphy - 12, neurotisation - 3, supplementary tenomioplasty - 23. Evaluation of the results with the use of BMRC scale (M0-M5) and Highet scale (S0-S4) included the group of 120 patients. After the surgical treatment a significant improvement was found in 63.3%. The efficacy of the treatment is strictly dependent on an early surgical intervention, mechanism and degree of the nerve injury as well as appropriate method of surgical therapy.

[Tumors and tumor-like lesions of eyelids collected at Department of Pathological Anatomy, Wroclaw Medical University, between 1946 and 1999]. / Nowotwory i zmiany nowotworopodobne powiek w materiale zakladu anatomii patologicznej akademii medycznej we wroclawiu w latach 1946-1999.

PURPOSE: To determine the histopathological, epidemiological and clinical characteristics of benign, malignant and tumor-like lesions of the eyelid collected in Department of Pathological Anatomy, Wroclaw Medical University. MATERIAL AND METHODS: Department of Pathological Anatomy, Wroclaw Medical University, has been collecting data of all tumors and tumor-like lesions of the eye and ocular adnexa diagnosed at the department during the 54-year interval from 1946 through 1999. Retrospective analysis of all eyelid biopsy specimens submitted to the department between January 1, 1946, and December 31, 1999 were performed. RESULTS: There were 4818 cases of the eye and ocular adnexa lesions diagnosed at the department since 1946 and 1999, what made up 0.262% of all diagnosed cases. Of 4818 total number of cases there were 2416 women and 2392 men. In 10 cases sex was unknown. The eyelids were the most common localization of malignant tumors (39.9% cases of all eye regions). The incidence cohort of all eyelid lesions included 2031 cases what made up 42.15% of all diagnosed lesions within the eye and it's adnexa. Benign tumors were the most common lesions (n=1262; 62.2%). Among all them papillomas were the most commonly found (n=659; 52.3%). Malignant tumors constituted 21,3% (n=433). Of the 433 malignancies, 314 were basal cell carcinomas (72.5%). Malignant tumors developed most commonly on the lower eyelid (n=303; 70%) but involved the right and left sides with equal frequency. Almost 2/3 of all tumor-like lesions in both genders were cysts including atheromas. CONCLUSIONS: Benign eyelid tumors compose the majority of all eyelid lesions. The eyelids are the most common localization of malignant tumors within the eye and it's adnexa. Basal cell carcinoma is the most common malignant eyelid tumor. The lower eyelid is the most frequent site of origin in cases of malignant tumors. Men and women are equally affected.

Brachial plexus tumours--own experience in diagnostics and surgical treatment.

Brachial plexus tumours are rather rare peripheral nerve tumours. In this study, we have presented the results of surgical treatment of cases with neoplasmatic process in the brachial plexus structures. Clinical material consisted of 9 patients (8 women, 34-72 years old; and 1 boy, 7 years old) on which the following procedures were performed: a) excision of the tumour with immediate nerve grafting of the sural nerve, b) extirpation of the tumour, c) excision of the tumour and direct suture reconstruction, d) brachial plexus neurolysis, e) tumour biopsy. We evaluated the pre- and post-operative motor and sensory deficit and established the three point scale of deficit intensity from + to +++. We diagnosed the following tumours in the clinical material: neurofibroma (diagnosed in 4 cases), plexiform neurofibroma (diagnosed in 2 cases), neurilemmoma (diagnosed in 1 case), and malignant tumours (diagnosed in 2 cases), which were found at the plexus area; they were metastatic or spreading continuously from the surrounding tissue. After surgical treatment, the sensory and motor function improved significantly in 4 cases (44,4%). The surgical treatment results depended on the size, localization and histological pattern of the tumour.

Influence of exogenous melatonin on doxorubicin-evoked effects in myocardium and in transplantable Morris hepatoma in rats.

BACKGROUND: Melatonin is one of the most effective oxygen free radical scavengers. Its cardio- and nephro-protective effects were demonstrated in the course of anthracycline administration. The present study aimed at examining the cytostatic efficiency of doxorubicin (DOX) applied together with melatonin to rats with transplantable Morris hepatoma. MATERIALS AND METHODS: Animals were divided into four groups receiving 0.9% NaCl, melatonin, DOX and melatonin together with DOX, respectively. Three weeks after transplantation of the tumour cells, the heart and tumour were isolated. The extent of tumour necrosis and the number of apoptotic cells were evaluated in paraffin-embedded sections. RESULTS: Administration of melatonin together with DOX decreased the extent of tumour necrosis and the apoptotic tumour cells but, on the other hand, decreased the number of apoptotic cardiomyocytes. CONCLUSION: Melatonin weakens the cytotoxic activity of DOX by the decreased proportions of necrotic and apoptotic cells of transplantable Morris hepatoma. Melatonin protects also cardiomyocytes by decreasing DOX-induced apoptosis in the cells.

Low-frequency electromagnetic stimulation may lead to regression of Morris hepatoma in buffalo rats.

OBJECTIVE: The influence of low-frequency electromagnetic (LF-EM) waves on the processes of carcinogenesis and tumor growth has been the subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting method of complementary medicine, biophysical-information therapy (BIT) or bioresonance therapy (BRT), which in principle is based on LF-EM stimulation, has emerged. BRT has been known since the late 1980s but is still poorly studied. The idea of applying BRT to tumors is based on two main premises: (1) endogenous biophotonic emission in the case of tumors is different from that produced by healthy tissues/cells and (2) BRT effects are believed to be primarily manifested at the immune-system level. Consequently, we decided to study the influence of BRT on a dynamic and well-known process: the expansion of transplantable hepatoma in rats. DESIGN: The study was carried out on female Buffalo rats with implanted Morris tumors (three experimental and one control group). Fourteen (14) consecutive in vivo exposures using a BRT device (BICOM B15, REGUMED Regulative Medizintechnik Gmbh, Graefelfing, Germany), were made from the third day after inoculation of the tumors. Biometric observations, intra vitam (tumor volume, growth ratio), were completed with histologic investigation (implantation locus, selected internal organs [lungs]). RESULTS: Thirty-one (31) cases (69%; n = 45) of total tumor regression were observed in experimental groups and these individuals were anesthetized to enable histologic verification to be made. No lung metastases--usually observed in tumor-bearers--could be detected. Moreover, in the inoculation loci, traces of former implantation and tumor absorption were found to be associated with high activity of cell-mediated immune response. No regressions were observed in the control group. CONCLUSIONS: We cannot exclude the possibility that LF-EM signals transmitted via BRT into the tumor-bearers may stimulate two separate processes: effective immunological response and/or tumor-cell death. The method appears to be capable of inducing the regression of transplantable hepatoma in vivo, thus is a potential subject of further studies.

Organ donor with cerebellar medulloblastoma: a case report and review of the literature.

Transplantation of organs and tissues procured from brain-dead organ donor (BDOD) is nowadays a common and desirable therapy for end-stag organ failure, especially kidneys, liver and heart. The majority of organs for transplantation are obtained from individuals with massive and irreversible central nervous system injury. This group of organ donors is however unsatisfactory and small according to needs. Significant percentage of multiorgan donors could be found among patients suffering from primary cerebral neoplasms as these rarely metastasize spontaneously outside the central nervous system. The paper presents the case of a 30-year-old male donor with a central nervous system tumor. Histogenesis, pathway of malignant dissemination, prognosis and the use of organ donor with cerebellar medulloblastoma are also discussed.