Broad and potent neutralizing antibodies are elicited in vaccinated individuals following Delta/BA.1 breakthrough infection.

IMPORTANCE: With the emergence of SARS-CoV-2 viral variants, there has been an increase in infections in vaccinated individuals. Here, we isolated monoclonal antibodies (mAbs) from individuals experiencing a breakthrough infection (Delta or BA.1) to determine how exposure to a heterologous Spike broadens the neutralizing antibody response at the monoclonal level. All mAbs isolated had reactivity to the Spike of the vaccine and infection variant. While many mAbs showed reduced neutralization of current circulating variants, we identified mAbs with broad and potent neutralization of BA.2.75.2, XBB, XBB.1.5, and BQ.1.1 indicating the presence of conserved epitopes on Spike. These results indicate that variant-based vaccine boosters have the potential to broaden the vaccine response.

A Wideband High-Gain Microstrip Array Antenna Integrated with Frequency-Selective Surface for Sub-6 GHz 5G Applications.

This paper presents a wideband and high-gain rectangular microstrip array antenna with a new frequency-selective surface (FSS) designed as a reflector for the sub-6 5G applications. The proposed antenna is designed to meet the US Federal Communications Commission (FCC) standard for 5G in the mid-band (3.5-5 GHz) applications. The designed antenna configuration consists of 1 × 4 rectangular microstrip array antenna with an FSS reflector to produce a semi-stable high radiation gain. The modeled FSS delivered a wide stopband transmission coefficient from 3.3 to 5.6 GHz and promised a linearly declining phase over the mid-band frequencies. An equivalent circuit (EC) model is additionally performed to verify the transmission coefficient of the proposed FSS structure for wideband signal propagation. A low-cost FR-4 substrate material was used to fabricate the antenna prototype. The proposed wideband array antenna with an FSS reflector attained a bandwidth of 2.3 GHz within the operating frequency range of 3.5-5.8 GHz, with a fractional bandwidth of 51.12%. A high gain of 12.4 dBi was obtained at 4.1 GHz with an improvement of 4.4 dBi compared to the antenna alone. The gain variation was only 1.0 dBi during the entire mid-band. The total dimension of the fabricated antenna prototype is 10.32 λo × 4.25 λo ×1.295 λo at a resonance frequency of 4.5 GHz. These results make the presented antenna appropriate for 5G sub-6 GHz applications.

Broad Neutralization of SARS-CoV-2 Variants, Including Omicron, following Breakthrough Infection with Delta in COVID-19-Vaccinated Individuals.

Numerous studies have shown that a prior SARS-CoV-2 infection can greatly enhance the antibody response to COVID-19 vaccination, with this so called "hybrid immunity" leading to greater neutralization breadth against SARS-CoV-2 variants of concern. However, little is known about how breakthrough infection (BTI) in COVID-19-vaccinated individuals will impact the magnitude and breadth of the neutralizing antibody response. Here, we compared neutralizing antibody responses between unvaccinated and COVID-19-double-vaccinated individuals (including both AZD1222 and BNT162b2 vaccinees) who have been infected with the Delta (B.1.617.2) variant. Rapid production of spike-reactive IgG was observed in the vaccinated group, providing evidence of effective vaccine priming. Overall, potent cross-neutralizing activity against current SARS-CoV-2 variants of concern was observed in the BTI group compared to the infection group, including neutralization of the Omicron (B.1.1.529) variant. This study provides important insights into population immunity where transmission levels remain high and in the context of new or emerging variants of concern. IMPORTANCE COVID-19 vaccines have been vital in controlling SARS-CoV-2 infections and reducing hospitalizations. However, breakthrough SARS-CoV-2 infections (BTI) occur in some vaccinated individuals. Here, we study how BTI impacts on the potency and the breadth of the neutralizing antibody response. We show that a Delta infection in COVID-19-vaccinated individuals provides potent neutralization against the current SARS-CoV-2 variants of concern, including the Omicron variant.

Humoral responses following SARS-CoV-2 breakthrough infection in COVID-19 vaccinated individuals

Numerous studies have shown that a prior SARS-CoV-2 infection can greatly enhance the antibody response to COVID-19 vaccination, with this so called "hybrid immunity" leading to greater neutralization breadth against SARS-CoV-2 variants of concern. However, little is known about how breakthrough infection (BTI) in COVID-19 vaccinated individuals will impact the magnitude and breadth of the neutralizing antibody response. Here, we compared neutralizing antibody responses between unvaccinated and COVID-19 double vaccinated individuals (including both AZD1222 and BNT162b2 vaccinees) who have been infected with the delta (B.1.617.2) variant. Rapid production of Spike-reactive IgG was observed in the vaccinated group providing evidence of effective vaccine priming. Overall, potent cross-neutralizing activity against current SARS-CoV-2 variants of concern was observed in the BTI group compared to the infection group, including neutralization of the omicron (B.1.1.529) variant. This study provides important insights into population immunity where transmission levels remain high and in the context of new or emerging variants of concern.

Functional capacities of microbial communities to carry out large scale geochemical processes are maintained during ex situ anaerobic incubation.

Mechanisms controlling CO2 and CH4 production in wetlands are central to understanding carbon cycling and greenhouse gas exchange. However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO2 and CH4 dynamics.

Cetuximab Conjugated with Octreotide and Entrapped Calcium Alginate-beads for Targeting Somatostatin Receptors.

There is a need to formulate oral cetuximab (CTX) for targeting colorectal cancer, which is reported to express somatostatin receptors (SSTRs). Therefore, coating CTX with a somatostatin analogue such as octreotide (OCT) is beneficial. Alginate was used to coat CTX to facilitate delivery to the gastrointestinal tract (GIT). This study aimed to deliver CTX conjugated with OCT in the form of microparticles as a GIT-targeted SSTR therapy. Both CTX and OCT were conjugated using a solvent evaporation method and the conjugated CTX-OCT was then loaded onto Ca-alginate-beads (CTX-OCT-Alg), which were characterized for drug interactions using differential scanning calorimetry (DSC), and Fourier transform infrared spectra (FTIR). Moreover, the morphology of formulated beads was examined using a scanning electron microscope (SEM). The drug content and release profile were studied using UV spectroscopy. Finally, in vitro cytotoxicity of all compounds was evaluated. The results showed homogenous conjugated CTX-OCT with a diameter of 0.4 mm. DSC showed a delay in the OCT peak that appeared after 200 °C due to small polymer interaction that shifted the OCT peak. Moreover, FTIR showed no prominent interaction. SEM showed clear empty cavities in the plain Ca-alginate-beads, while CTX-OCT-Alg showed occupied beads without cavities. CTX-OCT-Alg had a negligible release in 0.1 N HCl, while the CTX-OCT was completely released after 300 min in phosphate buffer pH 7.4. All formulations showed good antiproliferative activity compared with free drugs. The formulated CTX-OCT-Alg are a promising platform for targeting colorectal cancer through GIT.

Urinary megalin in association with progression factors of diabetic nephropathy.

AIM: The aim of this study is to evaluate the association between urinary megalin, renal function, blood pressure, lipid profile, vitamin D and glycemic control in patients with type 2 diabetes mellitus (T2DM). METHODS: . This was a cross-sectional study which recruited 209 patients with T2DM. Urinary megalin was positively associated with systolic blood pressure (SBP) (r=0.218, p=0.04) but negatively with glomerular filtration rate (GFR) (r=-0.16, p=0.023). The levels of urinary albumin, triglycerides (TGs) and glycosylated hemoglobin (HbA1c) were higher in the "high-megalin" group, compared to those in "low-megalin" group. Moreover, there was a significant inverse association between vitamin D3 levels and megalin levels in urine (OR=0.281, p=0.047). CONCLUSION: Our study showed for the first time that megalin is associated with progression factors of diabetic nephropathy as well as vitamin D deficiency (Tab. 3, Fig. 1, Ref. 15).

Cytotoxicity of propolis nanopreparations in cancer cell monolayers: multimode of action including apoptotsis and nitric oxide production.

Natural products are invaluable resource of anticancer drug discovery. They generally viewed as safe but weak, within the framework of nanotechnology, they can serve as template for potent anticancer drugs. We first evaluated the cytotoxic activity of different propolis extracts (water, 70% ethanol, absolute ethanol and hexane) in many cancer cell lines, then the solid nanoparticles from the organic solvent extracts were prepared and their cytotoxicity was evaluated as well. Finally, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes were prepared from the most cytotoxic organic solvent extract and their cytotoxicity was also evaluated. All results collectively showed that hexane extract and its solid nanoparticles as well as its liposomal form exhibited high cytotoxic activity. DPPC/DOPC-hexane extract cytotoxicity selectively depends on the cell line and DOPC liposomal form was characterized by reduced IC50 compared with the other preparations/extracts, the average IC50 value is 165.8 ± 3 µg/ml. The antiproliferative activity of propolis was associated to multiple modes of actions including apoptosis and nitric oxide production and as indicated by the HPLC and FTIR results, it is functioning in many propolis ingredients rather than a single component and influenced by the presence of more lipophilic components within the extract and not by the extract mass yield. These results may have an impact on the multidrug resistivity issue.

Morphogenesis of lingual papillae of one-humped camel (Camelus dromedarius) during prenatal life: A light and scanning electron microscopic study.

This study was made on 24 camel fetuses of crown-rump vertebral length (CVRL) ranging from 10.5 cm to 105 cm CVRL (94-352 days old). These camel fetuses were classified into three groups representing the three trimesters of prenatal life. During the first trimester (94-142 days), lingual papillae (circumvallate and lentiform papillae) were demonstrated on the lingual root, but lingual body and the apex were almost free of papillae except for some scattered epithelial projections especially near the lateral borders of the body. In the second trimester (152-229 days), the lentiform papillae covered the entire root of the tongue except for areas occupied by the circumvallate papillae. Taste buds with clear pores were observed for the first time in areas between the circumvallate gustatory furrow and surface epithelium of the tongue. In addition, short numerous filiform papillae were observed on the rostral part of the lingual body and the lateral parts of the apex. Fungiform papillae, however, were demonstrated amidst the filiform papillae. In this trimester, taste buds were also seen on the top of the fungiform papillae. In the third trimester (256-352 days), all lingual papillae were clearly demonstrated on the dorsum of the root, body and apex of the tongue. Both types of gustatory papillae (circumvallate and fungiform) had well-developed taste buds. Mechanical papillae (filiform and lentiform) were well developed. Lentiform papillae occupied most of the dorsal aspect of the Torus linguae; they were larger in size with semicircular apices. Filiform papillae, however, were numerous and demonstrated heavily on the lateral and rostral parts of the body as well as on the apex of the tongue.

Chronic exposure to neonicotinoids reduces honey bee health near corn crops.

Experiments linking neonicotinoids and declining bee health have been criticized for not simulating realistic exposure. Here we quantified the duration and magnitude of neonicotinoid exposure in Canada's corn-growing regions and used these data to design realistic experiments to investigate the effect of such insecticides on honey bees. Colonies near corn were naturally exposed to neonicotinoids for up to 4 months-the majority of the honey bee's active season. Realistic experiments showed that neonicotinoids increased worker mortality and were associated with declines in social immunity and increased queenlessness over time. We also discovered that the acute toxicity of neonicotinoids to honey bees doubles in the presence of a commonly encountered fungicide. Our work demonstrates that field-realistic exposure to neonicotinoids can reduce honey bee health in corn-growing regions.

Crimean-Congo hemorrhagic fever virus in livestock ticks and animal handler seroprevalence at an abattoir in Ghana.

BACKGROUND: Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a zoonotic virus transmitted by Ixodid ticks and causes Crimean-Congo hemorrhagic fever (CCHF) disease in humans with up to 50 % mortality rate. METHODS: Freshly slaughtered livestock at the Kumasi abattoir in the Ashanti Region of Ghana were examined for the presence of ticks once a month over a 6-month period from May to November 2011. The ticks were grouped into pools by species, sex, and animal source. CCHFV was detected in the ticks using reverse transcription PCR. Blood samples were collected from enrolled abattoir workers at initiation, and from those who reported fever in a preceding 30-day period during monthly visits 2-5 months after initiation. Six months after initiation, all participants who provided baseline samples were invited to provide blood samples. Serology was performed using enzyme linked immunosorbent assay (ELISA). Demographic and epidemiological data was also obtained from enrolled participants using a structured questionnaire. RESULTS: Of 428 freshly slaughtered animals comprising 130 sheep, 149 cattle, and 149 goats examined, 144 ticks belonging to the genera Ambylomma, Hyalomma and Boophilus were identified from 57 (13.3 %): 52 (34.9 %), 4 (3.1 %) and 1 (0.7 %) cattle, sheep and goat respectively. Of 97 tick pools tested, 5 pools comprising 1 pool of Hyalomma excavatum and 4 pools of Ambylomma variegatum, collected from cattle, were positive for CCHFV. Of 188 human serum samples collected from 108 abattoir workers, 7 (3.7 %) samples from 6 persons were anti-CCHF IgG positive with one of them also being CCHF IgM positive. The seroprevalence of CCHFV identified in this study was 5.7 %. CONCLUSIONS: This study detected human exposure to CCHF virus in slaughterhouse workers and also identified the CCHF virus in proven vectors (ticks) of Crimean Congo hemorrhagic fever in Ghana. The CCHFV was detected only in ticks collected from cattle, one of the livestock known to play a role in the amplification of the CCHF virus.

Pyrethroid Insecticide Resistance Mechanisms in the Adult Phlebotomus papatasi (Diptera: Psychodidae).

Phlebotomus papatasi is one of the most medically important sand fly species in the Old World, serving as a vector of Leishmania parasites and phleboviruses. Chemical control is still considered the most effective method for rapidly reducing populations of flying insects involved in vector-borne disease transmission, but is increasingly threatened by insecticide resistance in the target insect posing significant problems for entomologists responsible for control programs. This study was conducted to determine pyrethroid resistance mechanisms and the biological, physiological, and molecular impacts of resistance in Ph. papatasi, and to compare their resistance mechanisms against those reported for mosquitoes and other intensely studied dipterans. Field-collected Ph. papatasi from Aswan, Egypt, were subjected to sublethal doses of permethrin and reared as a resistant strain under laboratory conditions through 16 generations. Biological parameter observations of resistant Ph. papatasi revealed an association of resistance with productivity cost. Physiological analysis revealed that concentrations of oxidase and esterase enzymes increased in early generations of the resistant colony, and then subsided through the F16 generation to levels similar to those in a susceptible colony. The activity levels of acetylcholinesterase were higher in field-collected Ph. papatasi than in susceptible colony flies, but decreased significantly despite subsequent exposure to permethrin. The molecular search for gene mutations in the resistant strain of Ph. papatasi failed to identify any mutations common in pyrethroid-resistant mosquitoes. Our study revealed that the mechanism of pyrethroid resistance in sand flies is different than that in mosquitoes, at least at the genetic level.

Stabilization of Cu(I) for binding and calorimetric measurements in aqueous solution.

Conditions have been developed for the comproportionation reaction of Cu(2+) and copper metal to prepare aqueous solutions of Cu(+) that are stabilized from disproportionation by MeCN and other Cu(+)-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu(+) complexes (Cu(I)(MeCN)3(+), Cu(I)Me6Trien(+), Cu(I)(BCA)2(3-), Cu(I)(BCS)2(3-)), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu(+) interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu(+) with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu(+)-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu(+) due to its potential for participating in unquantifiable ternary interactions.

Re-laparotomy after caesarean deliveries: risk factors and how to avoid?

The aim of this study was to explore the risk factors for and how to avoid re-laparotomy after caesarean delivery and to present the experience of a university tertiary care referral hospital. The study comprised of 2,000 caesarean deliveries, among which re-laparotomy was needed in 18 patients (0.9). The study found that 16 of the 18 cases that needed re-laparotomy had previous caesarean sections (CS) and 9/18 had placenta praevia. The main indication for the procedure was internal haemorrhage (haemoperitoneum) (12/18, 66.6%). Significant haemoperitoneum of > 2 litres was reported in six cases (33.3%). Maternal mortality occurred in 3/18 (16.6%) patients. The uterus was preserved in most patients (15 patients, 83.3%). A total of 12 patients needed re-suturing of the uterine incision; 10 patients had bilateral uterine artery ligation; and four patients had bilateral internal iliac artery ligation. Uterine compression B-Lynch suturing was needed in five patients with uterine atony. Six patients (33.3%) were admitted to the intensive care unit (ICU) and were discharged well. Re-laparotomy after caesarean delivery has many risk factors leading to postoperative haemorrhage. Early signs, such as tachycardia and hypotension must be closely monitored to allow early intervention and to avoid morbidity and mortality related to late re-laparotomy.

Transforming growth factor-ß1 gene polymorphism in mycosis fungoides.

BACKGROUND: Dysregulation in transforming growth factor (TGF)-ß1 signalling pathways has been linked to cancer. AIM: To study the association between single nucleotide polymorphisms (SNPs) of the TGF-ß1 gene and mycosis fungoides (MF). METHODS: Using restriction fragment length polymorphism analysis, SNPs in the TGF-ß1 gene were studied in 55 patients with MF of different stages and in 100 apparently healthy controls. RESULTS: A significant difference was found between patients and controls in distribution of the different TGF-ß1 genotypes, with mutant forms (T/C, T/T) encountered significantly more often in patients with MF (P < 0.001). The heterozygous genotype (T/C) was significantly associated with patch stage MF, whereas the homozygous genotype (T/T) was significantly associated with tumour stage (stage IIb) MF (P = 0.001), although this study included only a small number of these patients. CONCLUSIONS: Mutant TGF-ß1 genotypes are significantly associated with MF in Egyptian patients, with the homozygous genotype (T/T) having a stronger association with tumour stage (stage IIb).

Pregnancy outcome in women with familial Mediterranean fever.

This study comprised 74 infertile patients with familial Mediterranean fever (FMF), among which 26 women became pregnant. Pregnancies were followed according to the standard antenatal follow-up protocol of high-risk pregnancies. The principal outcome measures were the termination of pregnancy and its upshot. This study comprised 74 infertile patients with FMF. Of the cases, 12 (16.22%) had antiphospholipid syndrome (APS); 16 patients (21.62%) had a history of previous abdominal/pelvic surgery, which might have been contributing to delay of conception; 66 patients (89.18%) were on drug therapy by corticosteroids, colchicines and other agents. A total of 10/22 patients were delivered by caesarean section for complicated pregnancies. Six of the newborns were positive for the MEFV gene. Favourable pregnancy outcome occurs in patients with FMF treated with colchicine before and after pregnancy. Neonatal outcome was similar to that expected in the general population.

Common and novel transcriptional routes to behavioral maturation in worker and male honey bees.

Worker honey bees (Apis mellifera) undergo a process of behavioral maturation leading to their transition from in-hive tasks to foraging--a process which is associated with profound transcriptional changes in the brain. Changes in brain gene expression observed during worker behavioral maturation could represent either a derived program underlying division of labor or a general program unrelated to sociality. Male bees (drones) undergo a process of behavioral maturation associated with the onset of mating flights, but do not partake in division of labor. Drones thus provide an excellent reference point for polarizing transcriptional changes associated with behavioral maturation in honey bees. We assayed the brain transcriptomes of adult drones and workers to compare and contrast differences associated with behavioral maturation in the two sexes. Both behavioral maturation and sex were associated with changes in expression of thousands of genes in the brain. Many genes involved in neuronal development, behavior, and the biosynthesis of neurotransmitters regulating the perception of reward showed sex-biased gene expression. Furthermore, most of the transcriptional changes associated with behavioral maturation were common to drones and workers, consistent with common genetic and physiological regulation. Our study suggests that there is a common behavioral maturation program that has been co-opted and modified to yield the different behavioral and cognitive phenotypes of worker and drone bees.

Sarcocystis dubeyi (Huong and Uggla, 1999) infection in water buffaloes (Bubalus bubalis) from Egypt.

Water buffaloes (Bubalus bubalis) are intermediate hosts for 4 species of Sarcocystis , i.e., Sarcocystis fusiformis and Sarcocystis buffalonis with cats as definitive hosts; Sarcocystis levinei with dogs as definitive hosts; and Sarcocystis dubeyi with an unknown definitive host but thought to be zoonotic. Currently, the latter species has been identified with certainty only from Vietnam. In the present study, sarcocysts of S. dubeyi are reported in 11 (30%) of 35 Egyptian water buffaloes from which the esophageal muscles were examined histologically. Sarcocysts were microscopic, measuring 180-250 × 70-110 µm in size. Ultrastructurally, the sarcocyst wall was 3.5-6.5 µm thick and had palisade-like villar protrusions which give it a striated appearance. The villar protrusions contained microtubules that were distributed along the whole villus. This is the first report of S. dubeyi from water buffaloes in Egypt.

Characterization of midgut and salivary gland proteins of Hyalomma dromedarii females controlled by entomopathogenic nematodes.

Biological control of hard ticks, Hyalomma dromedarii (Acari: Ixodidae) using entomopathogenic nematode of two families; Heterorhabditidae and Steinernematidae was studied. The protective effect of controlled ticks including haemolymph and haemocytes against these biological agents were also investigated. It was found that heterorhabditid strains cause a higher effect in biological control of engorged female H. dromedarii than those of stienernematid strains. It induced mortality rates ranged from 12-92% versus 4-88% for stienernematid strains. It was also found that these entomopathogenic nematodes can not reproduce within the exposed ticks. SDS-PAGE of proteins extracted from midguts and salivary glands infected with 4000 IJs tick(-1) separated 21 and 25 protein bands versus 13 and 19 protein bands from non-infected ones, respectively. It was concluded that entomopathogenic nematodes of family Heterorhabditidae proved to have a potential acaricidal effect in the control of hard ticks. Moreover, the controlled ticks released unknown proteins in their haemolymph that may promote the haemocytes to phagocyte the nematodes as a type of defense mechanism.

Prenatal development of murine gonads with special reference to germ cell differentiation: a morphological and immunohistochemical study.

The prenatal differentiation of male and female gonads of the mouse was investigated both morphologically and immunohistochemically. Sexual dimorphism could be detected as early as 12 days post-coitum (dpc) by the appearance of the primary elements of the tunica albuginea and positive immunoreactivity for anti-Muellerian hormone in the Sertoli cells of the male gonad. Male germ cells passed two waves of mitotic activity, a first wave between 12 and 14 dpc, which is followed by apoptosis of the old germ cell generation, and a second wave between 17 and 20 dpc. Oct-4 was expressed as a juxtanuclear ring in the cytoplasm of germ cells up to 17 dpc. Subsequently, it was down-regulated and completely disappeared in 20 dpc full-term fetuses. By contrast, M2A antigen revealed only a weak immunoreaction in some germ cells of 14 dpc gonads, but exhibited strong signals in all germ cells of 20 dpc full-term fetuses. Therefore, we postulate that, in the mouse, prenatal germ cells represent two populations: the first is immunopositive for Oct-4 and disappeared in full-term fetuses, whereas the second appeared in 14 dpc and is immunopositive for M2A antigen.