Benign multiple sclerosis: physical and cognitive impairment follow distinct evolutions.

BACKGROUND: Benign multiple sclerosis (BMS) definitions rely on physical disability level but do not account sufficiently for cognitive impairment which, however, is not rare. OBJECTIVE: To study the evolution of physical disability and cognitive performance of a group of patients with BMS followed at an University Hospital Multiple Sclerosis Center. METHODS: A consecutive sample of 24 BMS cases (diagnosis according to 2005 McDonald's criteria, relapsing-remitting course, disease duration ≥ 10 years, and expanded disability status scale [EDSS] score ≤ 2.0) and 13 sex- and age-matched non-BMS patients differing from BMS cases for having EDSS score 2.5-5.5 were included. Main outcome measures were as follows: (i) baseline and 5-year follow-up cognitive impairment defined as failure of at least two tests of the administered neuropsychological battery; (ii) EDSS score worsening defined as confirmed increase ≥ 1 point (or 0.5 point if baseline EDSS score = 5.5). RESULTS: At inclusion, BMS subjects were 41 ± 8 years old and had median EDSS score 1.5 (range 0-2), while non-BMS patients were 46 ± 8 years old and had median EDSS score 3.0 (2.5-5.5). At baseline 16% of patients in both groups were cognitively impaired. After 5 years, EDSS score worsened in 8% of BMS and 46% of non-BMS patients (P = 0.008), while the proportion of cognitively impaired subjects increased to 25% in both groups. CONCLUSIONS: Patients with BMS had better physical disability outcome at 5 years compared to non-BMS cases. However, cognitive impairment frequency and decline over time appeared similar. Neuropsychological assessment is essential in patients with BMS given the distinct pathways followed by disease progression in cognitive and physical domains.

Testing the diet-breadth trade-off hypothesis: differential regulation of novel plant secondary compounds by a specialist and a generalist herbivore.

Specialist herbivores are predicted to have evolved biotransformation pathways that can process large doses of secondary compounds from the plant species on which they specialize. It is hypothesized that this physiological specialization results in a trade-off such that specialists may be limited in ability to ingest novel plant secondary compounds (PSCs). In contrast, the generalist foraging strategy requires that herbivores alternate consumption of plant species and PSC types to reduce the possibility of over-ingestion of any particular PSC. The ability to behaviorally regulate is a key component of this strategy. These ideas underpin the prediction that in the face of novel PSCs, generalists should be better able to maintain body mass and avoid toxic consequences compared to specialists. We explored these predictions by comparing the feeding behavior of two herbivorous rodents: a juniper specialist, Neotoma stephensi, and a generalist, Neotoma albigula, fed diets with increasing concentrations of phenolic resin extracted from the creosote bush (Larrea tridentata), which produces a suite of PSCs novel to both species. The specialist lost more mass than the generalist during the 15-day trial. In addition, although the specialist and generalist both regulated phenolic resin intake by reducing meal size while on the highest resin concentration (4%), the generalist began to regulate intake on the 2% diet. The ability of the generalist to regulate intake at a lower PSC concentration may be the source of the generalist's performance advantage over the specialist. These data provide evidence for the hypothesis that the specialist's foraging strategy may result in behavioral as well as physiological trade-offs in the ability to consume novel PSCs.

Generalized epilepsy and mild intellectual disability associated with 13q34 deletion: A potential role for SOX1 and ARHGEF7.

Terminal deletions of long arm of chromosome 13 are rare and poorly characterized by cytogenetic studies, making for difficult genotype-phenotype correlations. We report two siblings presenting generalized epilepsy, intellectual disability, and genitourinary tract defects. Array CGH detected a 1.3 Mb deletion at 13q34; it contains two protein-coding genes, SOX1 and ARHGEF7, whose haploinsufficiency can contribute to the epileptic phenotype.

Association of B-chronic lymphocytic leukemia and acute myeloid leukemia.

A 62-year-old man presented with fatigue, pallor and mild weight loss. Laboratory studies showed Hb 7.6 g/dl, Hct 21.8%, WBC 108x10(9)/1, PLT 143x10(9)/1. At morphological examination, circulating cells appeared as 60% blasts and 40% lymphocytes, with smudge cells. A bone marrow aspirate showed infiltration by blasts (50%) and lymphocytes (40%); alpha-naphtyl-acetate esterase was positive in 90% of blasts, while myeloperoxidase was positive in 10%. The immunologic phenotype of blasts was characterized by the co-expression of CD13, CD33, CD14, CD4, CD15, CD64, CD117, HLA-DR, CD11b. Lymphocytes were characterized by a B-CLL immunophenotype: CD19+, CD5+, CD23+, CD20+(dim), FMC7+(dim), K light chain+(dim). Karyotype was normal and PCR assays for AML-ETO, CBFbeta-MYH11, PML-RARalpha, BCR-ABL and bcl-1/JH translocation were negative. Coexistence of CLL and AML with monoblastic features was diagnosed. Simultaneous appearance of CLL and AML has rarely been described and represents a peculiar biological phenomenon.

Treatment of post-traumatic hand Staphylococcus aureus osteomyelitis with oral linezolid.

The Authors report on the use of linezolid for the treatment of three patients with osteomyelitis. All three patients had post-traumatic multisensitive hand bone methicillin-susceptible Staphylococcus aureus osteomyelitis, which did not respond to antimicrobial regimens including drugs in vitro active against the isolated strains. Clinical cure and microbiologic eradication was obtained with oral linezolid in all three patients. Linezolid was well tolerated. Mild thrombocytopenia was observed in one patient at the end of the third week of treatment and it was promptly resolved after the discontinuation of linezolid. Linezolid minimum inhibitory concentrations (MICs) consisted of 2 mg/l for all three S. aureus isolates while the bactericidal activity in vitro was not present up to the linezolid concentration of 32 mg/l. In spite of a lack of in vitro bactericidal activity, linezolid was effective in curing the patients and eradicating the infection. Trough and peak plasma concentrations of linezolid were above the MICs of the isolates. These values ranged from 3.93 to 14.95 mg/l at trough and 5.03 to 25.91 mg/l at peak. The oral bioavailability, pharmacokinetic profile and antibacterial spectrum of linezolid make this oxazolidonone antimicrobial an attractive drug for the treatment of chronic osteomyelitis. Prolonged administration requires careful surveillance for side effects, until these complications are better understood.

Glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor differently modifies actin polymerization in neutrophils.

AIM: Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG-CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. MATERIALS AND METHODS: We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. RESULTS: Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. CONCLUSIONS: Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation.

An image processing workstation for automatic evaluation of human granulocyte motility.

Polymorphonuclear (PMN) motility is currently determined by a method using chemotactic chambers with micropore filters. PMN locomotive capacity is measured by evaluating the maximum distance travelled by the cells, or by counting the number of cells which have moved through the filter. Although in recent years some attempts to improve the analysis of chemotaxis data by computer-assisted systems have been made no technique has been shown capable of both measurement and correlation of these parameters in real time. In the present paper we describe an automated technique, based on a workstation capable of exploring microporous filters employed in chemotactic chambers and of measuring PMN motility in a reliable, reproducible, rapid manner, independent of the subjectivity of the operator. The measurement is carried out by custom software capable of undertaking computerized image analysis of microscopic fields acquired by a TV camera and of driving the motorized microscopic table. Depth of migration, cell distribution at each plane, correlation index of the random behaviour with the model described by a gaussian distribution and data about the patient or assay under study, are computed, displayed, stored in a data base and printed in real time.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes--a flow cytometric study.

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study.

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

Contemporaneous appearance, 18 years after allogeneic bone marrow transplantation, of myelodysplastic syndrome in the patient and the donor.

We report the case of the development of two different stages of the same clonal disorder in two patients sharing the same bone marrow due to a previous bone marrow allotransplant. The transplanted patient developed severe aplasia with myeloid blasts, different from those of the previously cured leukemia. Chimerism evaluated by microsatellite analyses confirmed a full donor phenotype. At the same time, the donor of the bone marrow transplantation developed a refractory anemia with excess blasts. We speculate on the presence of an undetectable pre-existing pathological clone in the transplanted bone marrow, which have evolved in the two patients.

Parabrachial nucleus lesions block taste and attenuate flavor preference and aversion conditioning in rats.

Rats with ibotenic acid lesions of the parabrachial nucleus (PBN) failed to learn a taste aversion induced by lithium chloride (LiCl) toxicosis. The same rats also did not learn to prefer a taste that was paired with intragastric (IG) carbohydrate infusions during 22 hr/day trials. The PBN-lesioned rats did learn to prefer a flavor (odor + taste) paired with the IG carbohydrate infusions over a different flavor paired with IG water. The PBN-lesioned rats also learned to avoid a flavor paired with IG LiCl infusions during 22 hr/day trials. The flavor preference and aversion, however, were less pronounced than those displayed by control rats. These data indicate that the PBN is essential for forming orosensory-viscerosensory associations when taste is the primary cue but is less critical when more complex flavor cues are available.

Flavor preferences conditioned by intragastric sugar infusions in rats: maltose is more reinforcing than sucrose.

Prior research indicates that glucose conditions much stronger flavor preferences in rats than does fructose. This could occur because intestinal absorption of fructose is much slower than that of glucose and because fructose malabsorption may have aversive consequences. Fructose absorption is facilitated when glucose is also present in the gut. The present study therefore compared the flavor conditioning effects of maltose (a glucose + glucose disaccharide) to those of sucrose (a glucose + fructose disaccharide). In Experiment 1, rats had different flavors paired with intragastric infusions of 32% maltose (CS+M), 32% sucrose (CS+S), and water (CS-) 23 h/day. In subsequent two-bottle tests, both CS+ solutions were strongly preferred to the CS-, but the CS+M was also preferred (78%) to the CS+S. Experiment 2A revealed that the rats also learned to prefer a CS+M to a CS+S when 16% sugar infusions were used. In Experiment 2B, the same rats preferred a flavor paired with 16% maltose to a flavor paired with 8% maltose. They did not reliably prefer a flavor paired with 16% sucrose to a flavor paired with 8% maltose. These results demonstrate that the postingestive actions of maltose are more reinforcing than those of sucrose. This indicates that fructose is less reinforcing than glucose even when malabsorption is not a factor. In contrast to their preference for the CS+M over the CS+S, the rats preferred sucrose to maltose when drinking the sugars by mouth. Therefore, sugar preferences mediated by oral taste receptors differ from those conditioned by postoral nutrient detectors.

Conditioned flavor avoidance, preference, and indifference produced by intragastric infusions of galactose, glucose, and fructose in rats.

The postingestive reinforcing and satiating effects of intragastric (i.g.) infusions of 16% galactose, glucose, and fructose were compared in adult female rats. In Experiment 1, food-restricted rats were trained to drink (30 min/day) flavored solutions (the CS+Gal and CS+Glu) paired with intragastric (i.g.) infusions of galactose and glucose; other flavors (the CS-) were paired with IG water infusions. In subsequent choice tests, the rats strongly preferred (91%) the CS+Glu to the CS-, but avoided the CS+Gal (21%) in favor of the CS-. In Experiment 2, the rats were trained with a CS+Fru paired with i.g. fructose infusions and a CS- paired with i.g. water. In the choice test they consumed similar amounts of CS+Fru and CS- (CS+Fru preference = 51%). In other choice tests they preferred the CS+Glu (>80%) to CS+Fru and CS+Gal, and the CS+Fru (81%) to CS+Gal. Satiation tests were performed in Experiment 3 by adapting the rats to drink a 3% sugar + 0.2% saccharin solution paired with i.g. water infusions (30 min/day). On different test days 16% sugar instead of water was infused. IG galactose, glucose, and fructose produced comparable reductions in sugar+saccharin intake in the first test session. These findings demonstrate that, while the three sugars had similar satiating effects, they differed substantially in their postingestive flavor conditioning effects. The glucose and fructose results confirm prior data indicating that only glucose generates potent postingestive reinforcing stimuli. The galactose-induced flavor avoidance indicates that this sugar has a negative postingestive consequence. This may be due to the slow and incomplete hepatic metabolism of this sugar in adult rats. Conceivably, galactose intolerance may contribute to the lactose avoidance in adult animals.

Flavor preferences conditioned by intragastric polycose in rats: more concentrated polycose is not always more reinforcing.

Prior studies have obtained conditioned preferences for flavors paired with intragastric (IG) infusions of Polycose (hydrolyzed starch) at concentrations of 1-32% over a different flavor paired with IG water. The present study determined if rats would also learn to prefer a flavor paired with concentrated Polycose infusion over a flavor paired with a more dilute Polycose infusion. In Experiment 1, adult female rats were food-deprived and trained during alternating one-bottle sessions (30 min/day) to associate one flavored solution (the CS + 8) with IG infusions of 8% Polycose, a second flavored solution (the CS + 16) with IG infusions of 16% Polycose, and a third flavored solution (the CS-) with IG water infusions. In subsequent choice tests, the rats displayed similar preferences for the CS + 8 and CS + 16 over the CS-, but preferred the CS + 16 to CS + 8 in a direct choice test. A similar preference pattern was obtained in 22 h/day tests with the rats nondeprived. In Experiment 2, new rats were similarly trained and tested but with CS + 16 and CS + 32 solutions paired with 16% and 32% Polycose infusions, respectively. The rats preferred both CS+ solutions over the CS- solution in the short- and long-term tests. However, the CS + 16 was preferred over the CS + 32 by the food-deprived rats in the short-term tests. The two CS+ solutions were equally preferred in the long-term tests with food ad lib. These and other findings indicate that the postingestive reinforcing action of Polycose increases as concentration increases from 1% to 16% but does not increase further, and may actually decrease, at a 32% concentration. The rapid satiating effect of concentrated carbohydrate solutions may limit their reinforcing consequences.

Naltrexone fails to block the acquisition or expression of a flavor preference conditioned by intragastric carbohydrate infusions.

The effects of naltrexone on the expression and acquisition of flavor preferences conditioned by the postingestive actions of carbohydrates were investigated. Food-restricted rats (Experiment 1) were given one-bottle training with one flavored saccharin solution (CS+) paired with intragastric (IG) infusions of 16% sucrose, and another flavored saccharin solution (CS-) paired with water infusions. In two-bottle tests CS+ was preferred to CS-, and naltrexone (1.0-5.0 mg/kg) reduced total intake but not CS+ preference. In Experiment 2 food-restricted rats that received naltrexone (0.1 or 1.0 mg/kg; NTX group) throughout one-bottle training consumed less CS+ and CS- than did saline-treated control rats. Yet, the NTX and control groups displayed similar CS+ preferences during two-bottle tests when treated with saline or naltrexone (0.1-5.0 mg/kg). In Experiment 3, rats were trained to accept more CS+ than CS- in one-bottle tests. Naltrexone (0.1-2.5 mg/kg) reduced the one-bottle intakes of both solutions, and the rats continued to consume more CS+ than CS-. We conclude that the opioid system modulates the consumption of flavored solutions, but is not critically involved in the acquisition or expression of flavor preferences conditioned by IG carbohydrate.

D1 but not D2 dopamine receptor antagonism blocks the acquisition of a flavor preference conditioned by intragastric carbohydrate infusions.

The effects of dopamine D1 (SCH23390) and D2 (raclopride) receptor antagonists on the acquisition and expressions of flavor preferences conditioned by the postingestive actions of sucrose were investigated. Food-restricted rats were trained in one-bottle sessions to associate one flavored saccharin solution (CS+) with intragastric (i.g.) infusions of 16% sucrose, and another flavored saccharin solution (CS-) with water infusions. Flavor preferences were then measured in two-bottle tests. In Experiment 1A, rats that received the D2 antagonist (raclopride, 200 nmol/kg; RAC group) throughout training consumed less CS+ and CS- than did saline-treated Control rats; a saline-treated Yoked group had its intake limited to that of the RAC group. All three groups displayed CS+ preferences during two-bottle tests when treated with saline or raclopride, except at doses that greatly suppressed intake. Experiment 1B obtained similar results with rats treated with 400 nmol/kg raclopride throughout training. In Experiment 2, rats that received the D1 antagonist (SCH23390, 200 nmol/kg; SCH group) throughout training consumed less CS+ and CS- than did saline-treated Control rats; a saline-treated Yoked group had its intake limited to that of the SCH group. Unlike the Control and Yoked groups, the SCH group failed to prefer the CS+ to the CS- in two bottle tests. SCH23390 treatment during two-bottle testing did not block CS+ preference in the Control or Yoked groups, except at doses that greatly suppressed intake. We conclude that D1, but not D2, dopamine receptors are critically involved in the acquisition of a sucrose-conditioned flavor preference, and both receptor subtypes have a more limited role in the expression of this preference.

Abnormal migratory activity of peripheral neutrophils from asthmatic patients and its modulation by inhaled glucocorticoids.

In order to investigate if bronchial asthma is associated with enhanced markers of activation in peripheral neutrophils, the migratory capacity of neutrophils in venous blood was measured by means of the Boyden chamber technique in 29 subjects with bronchial asthma of differing severity. Random migration (random motility), but not locomotion toward 10% Escherichia coli supernatant as chemoattractant (chemotaxis), was increased in asthmatic subjects with respect to 11 normal subjects (98 +/- 20 microns vs. 85 +/- 6 microns; p < 0.05). When asthmatic subjects were subdivided into groups of different disease severity, subjects with mild and mild to moderate asthma showed significantly higher values for random motility and chemotaxis than normal subjects; on the other hand, subjects with more severe disease showed the lowest values for migratory activity. No correlation was found between migratory activity and clinical findings of asthma, except for baseline FEV1 (% of the predicted value), which showed a slight but significant positive correlation with chemotaxis (r = 0.44, p < 0.05). Subjects with atopic or occupational asthma had higher values for migratory activity than subjects with nonallergic asthma. Thirteen asthmatic subjects repeated all evaluations after 1 month of treatment with high doses of inhaled glucocorticoids (beclomethasone dipropionate 1500 micrograms/day). Random motility (95 +/- 24 microns vs. 75 +/- 15 microns; p < 0.05) and chemotaxis (130 +/- 22 microns vs. 105 +/- 25 microns; p < 0.05) were significantly reduced after treatment, as well as the symptom score; on the other hand, symptom score but not bronchial hyperresponsiveness to methacholine challenge significantly changed.(ABSTRACT TRUNCATED AT 250 WORDS)

fMLP-induced CD11b/CD18 upregulation on neutrophils from patients with non-Hodgkin's lymphomas treated with recombinant human granulocyte colony-stimulating factor.

The effects of rhG-CSF administration on fMLP-induced neutrophil CD11b and CD18 upregulation were studied in nine patients suffering from intermediate and high grade non-Hodgkin's lymphomas. Blood samples were obtained before recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration and 24 hrs after rhGSF interruption. The growth factor was administered subcutaneously for five days in a dosage of 5 microg/Kg/day. Nine normal subjects were studied as controls. Five patients showed an impaired baseline CD11b and CD18 upregulation, which was corrected by rhG-CSF therapy. Four patients showed a normal baseline CD11b and CD18 upregulation, but this function was reduced by rhG-CSF therapy. All patients showed a normal baseline fMLP-induced luminol-enhanced chemiluminiscence and significantly increased chemiluminescence values after rhG-CSF administration. We conclude that, while in some patients rhg-CSF is able to improve neutrophil CD11b and CD18 upregulation in response to chemotactic agents, in other patients a decrease of this function can occur, maybe due to a relative immaturity of the circulating neutrophils induced by rhG-CSF.

Inhibition of leukocyte function by serum from patients with trichinellosis.

Modification of leukocytic function has been reported in only a few human parasitic diseases. In this study we evaluated the effects of the sera from patients infected with Trichinella on chemotactic and phagocytic responses in leukocytes. Leukocyte chemotaxis was tested by the agarose method and phagocytosis by the technique of Yamamura, modified for Saccharomyces cerevisiae. Sera were acquired from patients during a trichinellosis outbreak that occurred in northern Italy in 1986. The parasite was isolated from 1 patient and isoenzymatically typed as Trichinella sp. 3, a new taxon, previously considered Trichinella nelsoni. The results indicated that sera from Trichinella-infected humans inhibited both chemotaxis and phagocytic responses in leukocytes. These findings suggest the existence of serum factor(s) in trichinellosis patients that modify host leukocytic functions. The source and nature of active serum components and the mechanism by which they modulate leukocyte function remain to be clarified.

Effects of lysine-arginine association on immune functions in patients with recurrent infections.

Combined L-lysine-L-arginine therapy is capable of inducting recovery in age-related decline of thymic activity in mice and in elderly humans. The clinical usefulness of the association has also been shown in children with recurrent respiratory infections, while an increase in the number of CD3+ lymphocytes has been shown in patients with chronic lymphatic leukaemia. Recently, in vitro effects of the association on neutrophil function have been reported. In particular, the association was able to increase random migration, chemotaxis, phagocytosis-associated- and f-MLP-induced chemiluminescence. In this paper the authors evaluate the effects of L-lysine-L-arginine combination (lisargin) on several humoral and cell-mediated immunologic parameters in patients with recurrent infection. An increase of neutrophil random migration and chemotaxis (evaluated by a new technique, based on a computer assisted image processing system) was found. Furthermore an increase in the absolute number of lymphocytes involved in cytotoxic activity and IgG levels was observed.